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Sample records for affinity chromatography sds-page

  1. Rapid discovery of putative protein biomarkers of traumatic brain injury by SDS-PAGE-capillary liquid chromatography-tandem mass spectrometry.

    PubMed

    Haskins, William E; Kobeissy, Firas H; Wolper, Regina A; Ottens, Andrew K; Kitlen, Jason W; McClung, Scott H; O'Steen, Barbara E; Chow, Marjorie M; Pineda, Jose A; Denslow, Nancy D; Hayes, Ronald L; Wang, Kevin K W

    2005-06-01

    We report the rapid discovery of putative protein biomarkers of traumatic brain injury (TBI) by SDS-PAGE-capillary liquid chromatography-tandem mass spectrometry (SDS-PAGE-Capillary LC-MS(2)). Ipsilateral hippocampus (IH) samples were collected from naive rats and rats subjected to controlled cortical impact (a rodent model of TBI). Protein database searching with 15,558 uninterpreted MS(2) spectra, collected in 3 days via data-dependent capillary LC-MS(2) of pooled cyanine dye-labeled samples separated by SDS-PAGE, identified more than 306 unique proteins. Differential proteomic analysis revealed differences in protein sequence coverage for 170 mammalian proteins (57 in naive only, 74 in injured only, and 39 of 64 in both), suggesting these are putative biomarkers of TBI. Confidence in our results was obtained by the presence of several known biomarkers of TBI (including alphaII-spectrin, brain creatine kinase, and neuron-specific enolase) in our data set. These results show that SDS-PAGE prior to in vitro proteolysis and capillary LC-MS(2) is a promising strategy for the rapid discovery of putative protein biomarkers associated with a specific physiological state (i.e., TBI) without a priori knowledge of the molecules involved. PMID:15941373

  2. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  3. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  4. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

    Describes the nature of affinity chromatography and its use in purifying enzymes, studying cell interactions, exploring hormone receptors, and other areas. The potential the technique may have in treating disease is also considered. (JN)

  5. Prestaining of glycoproteins in SDS-PAGE via 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide with weak influence on protein mobility.

    PubMed

    Zhu, Zhongxin; Zhou, Xuan; Wang, Yang; Yu, Qing; Zhu, Xinliang; Niu, Chao; Cong, Weitao; Jin, Litai

    2014-12-01

    A new fluorescent prestaining method for gel-separated glycoproteins in 1D and 2D SDS-PAGE was developed by using 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH). The prestained gels were readily imaged after electrophoresis without any time-consuming steps needed for poststain. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be selectively detected, which is comparable to the most commonly used Pro-Q Emerald 488 glycoprotein stain. In addition, subsequent study of deglycosylation, glycoprotein affinity chromatography, and LC-MS/MS analysis were performed to confirm the specificity of the newly developed method. As a result, BH prestain provides a new choice for quick, sensitive, specific, economical, and MS compatible visualization of gel-separated glycoproteins. PMID:25229714

  6. Affinity chromatography: a historical perspective.

    PubMed

    Hage, David S; Matsuda, Ryan

    2015-01-01

    Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods. PMID:25749941

  7. Detecting the mu opioid receptor in brain following SDS-PAGE with multiple approaches

    PubMed Central

    Huang, Peng; Liu-Chen, Lee-Yuan

    2013-01-01

    In general, it has been difficult to obtain antibodies which are useful for immunoblotting of endogenous seven-transmembrane receptors (7TMRs) despite the claims made by many companies on commercially available antibodies. In this review, we will use the mu opioid receptor (MOPR) in brain as an example to underscore the importance of using knock-out (K/O) mice and multiple independent approaches (ligand affinity-labeling, receptor phosphorylation and immunoblotting) in identifying 7TMRs following sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). The rigor and convergence of pharmacological and biochemical data provide confidence on the unequivocal identification of MOPR. The distinct relative molecular masses (Mr’s) and band patterns are largely due to variations in the extent of N-glycosylation in different cell lines, brain regions and species. PMID:19482639

  8. Determination of the Subunit Molecular Mass and Composition of Alcohol Dehydrogenase by SDS-PAGE

    ERIC Educational Resources Information Center

    Nash, Barbara T.

    2007-01-01

    SDS-PAGE is a simple, rapid technique that has many uses in biochemistry and is readily adaptable to the undergraduate laboratory. It is, however, a technique prone to several types of procedural pitfalls. This article describes the use of SDS-PAGE to determine the subunit molecular mass and composition of yeast alcohol dehydrogenase employing…

  9. Production and Purification of Streptokinase by Protected Affinity Chromatography

    PubMed Central

    Babashamsi, Mohammad; Razavian, Mohammad Hossein; Nejadmoghaddam, Mohammad Reza

    2009-01-01

    Streptokinase is an extracellular protein, extracted from certain strains of beta hemolytic streptococcus. It is a non-protease plasminogen activator that activates plasminogen to plasmin, the enzyme that degrades fibrin cloth through its specific lysine binding site; it is used therefore as a drug in thrombolytic therapy. The rate of bacterial growth and streptokinase production was studied in condition of excess glucose addition to culture media and its pH maintenance. The streptokinase product of the bacterial culture was preliminary extracted by salt precipitation and then purified by affinity chromatography on plasminogen substituted sepharose-4B in a condition that the plasminogen active site was protected from streptokinase-induced activation. The purity of streptokinase was confirmed by SDS-PAGE and its biological activity determined in a specific streptokinase assay. The results showed that in the fed–batch culture, the rate of streptokinase production increased over two times as compared with the batch culture while at the same time, shortening the streptokinase purification to a single step increased the yield over 95% at the chromatography stage. PMID:23407807

  10. Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose.

    PubMed

    DiScipio, Richard G; Liddington, Robert C; Schraufstatter, Ingrid U

    2016-05-01

    A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

  11. Lectin affinity chromatography of glycolipids

    SciTech Connect

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  12. Immobilized metal ion affinity chromatography.

    PubMed

    Yip, T T; Hutchens, T W

    1992-01-01

    Immobilized metal ion affinity chromatography (IMAC) (1,2) is also referred to as metal chelate chromatography, metal ion interaction chromatography, and ligand-exchange chromatography. We view this affinity separation technique as an intermediate between highly specific, high-affinity bioaffinity separation methods, and wider spectrum, low-specificity adsorption methods, such as ion exchange. The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups (e.g., His residues) in peptides (e.g., 3-7) and on protein surfaces (8-13). The number of stationary phases that can be synthesized for efficient chelation of metal ions is unlimited, but the critical consideration is that there must be enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. Several examples are presented in Fig. 1. The challenge to produce new immobilized chelating groups, including protein surface metal-binding domains (14,15) is being explored continuously. Table 1 presents a list of published procedures for the synthesis and use of stationary phases with immobilized chelating groups. This is by no means exhaustive, and is intended only to give an idea of the scope and versatility of IMAC. Fig. 1 Schematic illustration of several types of immobilized metal-chelating groups, including, iminodiacetate (IDA), tris(carboxymethyl) ethylenediamine (TED), and the metal-binding peptides (GHHPH)(n)G (where n = 1,2,3, and 5) (14,15). Table 1 Immobilized Chelating Groups and Metal Ions Used for Immobilized Metal Ion Affinity Chromatography Chelating group Suitable metal ions Reference Commercial source Immodiacetate Transitional1,2 Pharmacia LKB Pierce Sigma Boehringer Mannheim TosoHaas 2-Hydroxy-3[N-(2- pyrtdylmethyl) glycme]propyl Transitional3 Not available ?-Alky1 mtrilo triacetic acid Transitional4 Not available Carboxymethylated asparhc acid Ca(II)13 Not available Tris (carboxy- methyl) ethylene Diamme

  13. Affinity chromatography purification of angiotensin II reactor using photoactivable biotinylated probes

    SciTech Connect

    Marie, J.; Seyer, R.; Lombard, C.; Desarnaud, F.; Aumelas, A.; Jard, A.; Bonnafous, J.C. )

    1990-09-25

    The authors have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH{sub 2}){sub 2}SS(CH{sub 2}){sub 2}CO-(Ala{sup 1}, Phe(4N{sub 3}){sup 8})AII, which contains a cleavage disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (K{sub d}{approximately}1 nM), proved to be suitable for indirect affinity chromatography of rate liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors.

  14. One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE).

    PubMed

    Brunelle, Julie L; Green, Rachel

    2014-01-01

    This protocol describes a denaturing polyacrylamide gel system utilizing sodium dodecyl sulfate (SDS) to separate protein molecules based on size as first described by Laemmli (1970). SDS-PAGE can be used to monitor protein purifications, check the purity of samples, and to estimate molecular weights for unknown proteins. PMID:24674069

  15. Pre-staining of glycoprotein in SDS-PAGE by the synthesis of a new hydrazide derivative.

    PubMed

    Zhou, Ayi; Zhou, Tieli; Yu, Dongdong; Shen, Yingjie; Shen, Jiayi; Zhu, Zhongxin; Jin, Litai; Zhang, Huajie; Wang, Yang

    2015-11-01

    In this study, a new hydrazide derivative (UGF202) was synthesized and introduced as a highly sensitive and selective fluorescent probe to pre-stain glycoproteins in 1D and 2D SDS-PAGE. As low as 0.5-1 ng glycoproteins (transferrin, α1-acid glycoprotein, avidin) could be selectively detected, which is comparable to that of Pro-Q Emerald 300 stain, one of the most sensitive and commonly used glycoprotein staining kit. In addition, the specificity of the newly developed method was confirmed by the study of de-glycosylation, glycoproteins affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that UGF202 pre-stain can provide an alternative for the visualization of gel-separated glycoproteins. PMID:26256282

  16. Improved conditions for periodate/Schiff's base-based fluorescent staining of glycoproteins with dansylhydrazine in SDS-PAGE.

    PubMed

    Zhou, Xuan; Hong, Guo-Ying; Huang, Bin-Bin; Duan, Yuan-Meng; Shen, Jia-Yi; Ni, Mao-Wei; Cong, Wei-Tao; Jin, Li-Tai

    2014-05-01

    An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS-PAGE was described. Down to 4-8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16-fold higher than that of original protocol, but similar to that of Pro-Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel-separated glycoproteins. PMID:24591039

  17. Quantification of major allergen parvalbumin in 22 species of fish by SDS-PAGE.

    PubMed

    Kobayashi, Yukihiro; Yang, Tao; Yu, Cheng-Tao; Ume, Chiaki; Kubota, Hiroyuki; Shimakura, Kuniyoshi; Shiomi, Kazuo; Hamada-Sato, Naoko

    2016-03-01

    Fish is an important causative material of food allergy. Although the allergenicity of fish is considered to correlate with the content of parvalbumin, the major fish allergen, available information about the parvalbumin content in fish is limited. In this study, a simple and reliable quantification method for fish parvalbumin by SDS-PAGE was first established. Application of the SDS-PAGE method to 22 species of fish revealed a marked variation in parvalbumin content among fish. Furthermore, the parvalbumin content was found to be higher in dorsal white muscle than in ventral white muscle, in rostral part of white muscle than in caudal part of white muscle and in white muscle than in dark muscle. IgE reactivity of fish was roughly proportional to parvalbumin content. Interestingly, large-sized migratory fish, such as salmon, swordfish and tuna, were commonly very low in both parvalbumin content and IgE reactivity. PMID:26471564

  18. Purification of Bovine Carbonic Anhydrase by Affinity Chromatography: An Undergraduate Biochemistry Laboratory Experiment

    NASA Astrophysics Data System (ADS)

    Bering, C. Larry; Kuhns, Jennifer J.; Rowlett, Roger

    1998-08-01

    We have developed a rapid and inexpensive experiment utilizing affinity chromatography to isolate carbonic anhydrase (CA) from bovine blood. The more specific an affinity gel is the better the purification, but the greater the cost. Some costs would be prohibitive in the undergraduate biochemistry laboratory. Less specific resins may be more affordable but may bind a number of closely related proteins. One alternative would be to couple a specific ligand to an inexpensive resin such as an ion exchanger. We describe a simple procedure for preparing a sulfonamide-coupled resin which specifically binds CA from a blood hemolysate. The CA is eluted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was found that only a single band of 31 kD was obtained. The instructor can readily prepare the affinity gel prior to the lab, and the students, beginning with packed red blood cells can carry out the lysis, binding to the gel, elution, enzymatic assays, and electrophoresis.

  19. Loss of PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein.

    PubMed

    Zhang, Chun; Liu, Yongdong; Feng, Cui; Wang, Qi; Shi, Hong; Zhao, Dawei; Yu, Rong; Su, Zhiguo

    2015-01-01

    SDS-PAGE represents a quick and simple method for qualitative and quantitative analysis of protein and protein-containing conjugates, mostly pegylated proteins. PEG-maleimide (MAL) is frequently used to site-specifically pegylate therapeutic proteins via free cysteine residue by forming a thiosuccinimide structure for pursuing homogeneous products. The C-S linkage between protein and PEG-MAL is generally thought to be relatively stable. However, loss of intact PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein was observed. It is a thiol-independent thioether cleavage and the shedding of PEG chain exclusively happens to PEG-MAL modified conjugates although PEG-vinylsulfone conjugates to thiol-containing proteins also through a C-S linkage. Cleavage kinetics of PEG40k-MAL modified ciliary neurotrophic factor showed this kind of degradation could immediately happen even in 1 min incubation at high temperature and could be detected at physiological temperature and pH, although the rate was relatively slow. This may provide another degradation route for maleimide-thiol conjugate irrespective of reactive thiol, although the specific mechanism is still not very clear for us. It would also offer a basis for accurate characterization of PEG-MAL modified protein/peptide by SDS-PAGE analysis. PMID:25265901

  20. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography

    PubMed Central

    Lu, Lina; Qi, Zhijun; Zhang, Jiwen; Wu, Wenjun

    2015-01-01

    Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase) were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed. PMID:25996604

  1. Separation of Binding Protein of Celangulin V from the Midgut of Mythimna separata Walker by Affinity Chromatography.

    PubMed

    Lu, Lina; Qi, Zhijun; Zhang, Jiwen; Wu, Wenjun

    2015-05-01

    Celangulin V, an insecticidal compound isolated from the root bark of Chinese bittersweet, can affect the digestive system of insects. However, the mechanism of how Celangulin V induces a series of symptoms is still unknown. In this study, affinity chromatography was conducted through coupling of Celangulin V-6-aminoacetic acid ester to the CNBr-activated Sepharose 4B. SDS-PAGE was used to analyze the collected fraction eluted by Celangulin V. Eight binding proteins (Zinc finger protein, Thioredoxin peroxidase (TPx), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SUMO E3 ligase RanBP2, Transmembrane protein 1, Actin, APN and V-ATPase) were obtained and identified by LC/Q-TOF-MS from the midgut of Mythimna separata larvae. The potential of these proteins to serve as target proteins involved in the insecticidal activity of Celangulin V is discussed. PMID:25996604

  2. Genetic Variability among Lucerne Cultivars Based on Biochemical (SDS-PAGE) and Morphological Markers

    NASA Astrophysics Data System (ADS)

    Farshadfar, M.; Farshadfar, E.

    The present research was conducted to determine the genetic variability of 18 Lucerne cultivars, based on morphological and biochemical markers. The traits studied were plant height, tiller number, biomass, dry yield, dry yield/biomass, dry leaf/dry yield, macro and micro elements, crude protein, dry matter, crude fiber and ash percentage and SDS- PAGE in seed and leaf samples. Field experiments included 18 plots of two meter rows. Data based on morphological, chemical and SDS-PAGE markers were analyzed using SPSSWIN soft ware and the multivariate statistical procedures: cluster analysis (UPGMA), principal component. Analysis of analysis of variance and mean comparison for morphological traits reflected significant differences among genotypes. Genotype 13 and 15 had the greatest values for most traits. The Genotypic Coefficient of Variation (GCV), Phenotypic Coefficient of Variation (PCV) and Heritability (Hb) parameters for different characters raged from 12.49 to 26.58% for PCV, hence the GCV ranged from 6.84 to 18.84%. The greatest value of Hb was 0.94 for stem number. Lucerne genotypes could be classified, based on morphological traits, into four clusters and 94% of the variance among the genotypes was explained by two PCAs: Based on chemical traits they were classified into five groups and 73.492% of variance was explained by four principal components: Dry matter, protein, fiber, P, K, Na, Mg and Zn had higher variance. Genotypes based on the SDS-PAGE patterns all genotypes were classified into three clusters. The greatest genetic distance was between cultivar 10 and others, therefore they would be suitable parent in a breeding program.

  3. SDS-PAGE and two-dimensional maps in a radial gel format.

    PubMed

    Millioni, Renato; Miuzzo, Manuela; Antonioli, Paolo; Sbrignadello, Stefano; Iori, Elisabetta; Dosselli, Ryan; Puricelli, Lucia; Kolbe, Markus; Tessari, Paolo; Righetti, Pier Giorgio

    2010-01-01

    A novel method for performing 2-D map analysis is here reported, consisting in a modification of the second dimension run, which is performed not in a conventional square- or rectangular-size gel, but in a radial surface. This has the advantage of permitting resolution of closely adjacent bands, representing strings of isoforms of similar or identical mass but of closely spaced isoelectric points. When used in a mono-dimensional, SDS-PAGE format, this system allows the simultaneous running of 62 sample tracks. Examples are given of separation of plasma and urinary proteins. PMID:20119955

  4. Detection of an endothelin-1-binding protein complex by low temperature SDS-PAGE

    SciTech Connect

    Takasuka, T.; Horii, I.; Furuichi, Y.; Watanabe, T. )

    1991-04-15

    We found that the complex of ET-1 and its binding protein was stable enough to be separated by SDS-PAGE when electrophoresis was run at a low temperature. Cross-linking was not necessary for the detection of {sup 125}I-ET-1 and its binding protein complex by autoradiography. This simple method could be used in qualitative (estimation of apparent molecular weight of ET-1 binding protein) and quantitative (determination of relative content of ET-binding protein) analysis of the ET-binding protein complex. ET-binding protein complexes of various animal species and organs were investigated by this method.

  5. An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide.

    PubMed

    Guan, Yihong; Zhu, Qinfang; Huang, Delai; Zhao, Shuyi; Jan Lo, Li; Peng, Jinrong

    2015-01-01

    The molecular weight (MW) of a protein can be predicted based on its amino acids (AA) composition. However, in many cases a non-chemically modified protein shows an SDS PAGE-displayed MW larger than its predicted size. Some reports linked this fact to high content of acidic AA in the protein. However, the exact relationship between the acidic AA composition and the SDS PAGE-displayed MW is not established. Zebrafish nucleolar protein Def is composed of 753 AA and shows an SDS PAGE-displayed MW approximately 13 kDa larger than its predicted MW. The first 188 AA in Def is defined by a glutamate-rich region containing ~35.6% of acidic AA. In this report, we analyzed the relationship between the SDS PAGE-displayed MW of thirteen peptides derived from Def and the AA composition in each peptide. We found that the difference between the predicted and SDS PAGE-displayed MW showed a linear correlation with the percentage of acidic AA that fits the equation y = 276.5x - 31.33 (x represents the percentage of acidic AA, 11.4% ≤ x ≤ 51.1%; y represents the average ΔMW per AA). We demonstrated that this equation could be applied to predict the SDS PAGE-displayed MW for thirteen different natural acidic proteins. PMID:26311515

  6. Statistical theory of chromatography: new outlooks for affinity chromatography.

    PubMed Central

    Denizot, F C; Delaage, M A

    1975-01-01

    We have developed further the statistical approach to chromatography initiated by Giddings and Eyring, and applied it to affinity chromatography. By means of a convenient expression of moments the convergence towards the Laplace-Gauss distribution has been established. The Gaussian character is not preserved if other causes of dispersion are taken into account, but expressions of moments can be obtained in a generalized form. A simple procedure is deduced for expressing the fundamental constants of the model in terms of purely experimental quantities. Thus, affinity chromatography can be used to determine rate constants of association and dissociation in a range considered as the domain of the stopped-flow methods. PMID:1061072

  7. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-01-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands. PMID:25829303

  8. Analysis of the Glycoproteome of Toxoplasma gondii using Lectin Affinity Chromatography and Tandem Mass Spectrometry

    PubMed Central

    Luo, Qilie; Upadhya, Rajendra; Zhang, Hong; Madrid-Aliste, Carlos; Nieves, Edward; Kim, Kami; Angeletti, Ruth Hogue; Weiss, Louis M.

    2011-01-01

    Glycoproteins are involved in many important molecular recognition processes including invasion, adhesion, differentiation, and development. To identify the glycoproteins of Toxoplasma gondii, a proteomic analysis was undertaken. T. gondii proteins were prepared and fractioned using lectin affinity chromatography. The proteins in each fraction were then separated using SDS-PAGE and identified by tryptic in gel digestion followed by tandem mass spectrometry. Utilizing these methods 132 proteins were identified. Among the identified proteins were 17 surface proteins, 9 microneme proteins, 15 rhoptry proteins, 11 heat shock proteins (HSP), and 32 hypothetical proteins. Several proteins had 1 to 5 transmembrane domains (TMD) with some being as large as 608.3 kDa. Both lectin-fluorescence labeling and lectin blotting were employed to confirm the presence of carbohydrates on the surface or cytoplasm of T. gondii parasites. PCR demonstrated that selected hypothetical proteins were expressed in T. gondii tachyzoites. This is data provides a large scale analysis of the T. gondii glycoproteome. Studies of the function of glycosylation of these proteins may help elucidate mechanism(s) involved in invasion improving drug therapy as well as identify glycoproteins that may prove to be useful as vaccine candidates. PMID:21920448

  9. Displacement phenomena in lectin affinity chromatography.

    PubMed

    Cho, Wonryeon

    2015-10-01

    The work described here examines displacement phenomena that play a role in lectin affinity chromatography and their potential to impact reproducibility. This was achieved using Lycopersicon esculentum lectin (LEL), a lectin widely used in monitoring cancer. Four small identical LEL columns were coupled in series to form a single affinity chromatography system with the last in the series connected to an absorbance detector. The serial affinity column set (SACS) was then loaded with human plasma proteins. At the completion of loading, the column set was disassembled, the four columns were eluted individually, the captured proteins were trypsin digested, the peptides were deglycosylated with PNGase F, and the parent proteins were identified through mass spectral analyses. Significantly different sets of glycoproteins were selected by each column, some proteins appearing to be exclusively bound to the first column while others were bound further along in the series. Clearly, sample displacement chromatography (SDC) occurs. Glycoproteins were bound at different places in the column train, identifying the presence of glycoforms with different affinity on a single glycoprotein. It is not possible to see these phenomena in the single column mode of chromatography. Moreover, low abundance proteins were enriched, which facilitates detection. The great advantage of this method is that it differentiates between glycoproteins on the basis of their binding affinity. Displacement phenomena are concluded to be a significant component of the separation mechanism in heavily loaded lectin affinity chromatography columns. This further suggests that care must be exercised in sample loading of lectin columns to prevent analyte displacement with nonretained proteins. PMID:26348026

  10. Grain protein variability among species of Triticum and Aegilops: quantitative SDS-PAGE studies.

    PubMed

    Cole, E W; Fullington, J G; Kasarda, D D

    1981-01-01

    Total proteins were extracted from degermed seeds of various species of Triticum and Aegilops with solutions containing sodium dodecyl sulfate (SDS) and mercaptoethanol. The reduced, dissociated proteins were fractionated according to molecular weight (MW) by high-resolution polyacrylamide gel electrophoresis in buffers containing SDS (SDS-PAGE). Stained SDS-PAGE patterns were measured by densitometric scanning over a suitable range of optical density. The data were normalized to equivalent total areas for each of the densitometric scans by means of a computer program that also permitted the construction of patterns of hypothetical amphiploids by averaging patterns of two or three diploid species. The grain proteins of most species examined had distinctive qualitative and quantitative aspects that were characteristic of the species even though nearly every accession or cultivar of a species exhibited at least minor differences in pattern from other accessions or cultivars. The main protein components (probably prolamins) of Triticum monococcum ssp. monococcum, T. monococcum ssp. boeoticum, T. urartu, and Aegilops squarrosa had MW's in the range 29-36 X 10(3) whereas the most important components of Ae. speltoides, Ae. longissima, and Ae. searsii had MW's in the range 37-55 × 10(3). Changes in the quantitative expression of particular genes, especially those coding for storage protein components, may have been associated with speciation. The strong predominance of proteins with MW's in the range 29-36 × 10(3) in some accessions of AB genome tetraploids, such as T. turgidum ssp. dicoccoides, may indicate contributions to the B genome of these tetraploids by T. monococcum ssp. boeoticum, T. urartu, or Ae. squarrosa. PMID:24276584

  11. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of urinary protein in acute kidney injury.

    PubMed

    Suhail, Sufi M; Woo, K T; Tan, H K; Wong, K S

    2011-07-01

    Recent experimental and clinical studies have shown the importance of urinary proteomics in acute kidney injury (AKI). We analyzed the protein in urine of patients with clinical AKI using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for its diagnostic value, and followed them up for 40 months to evaluate prognosis. Urine from 31 consecutive cases of AKI was analyzed with SDS-PAGE to determine the low, middle and high molecular weight proteins. Fractional excretion of sodium (FENa) was estimated from serum and urine creatinine and sodium (Na). The cases were followed-up for 40 months from the end of the recruitment of study cases. Glomerular protein was higher in the hematuria group when compared with the non-hematuria group (P <0.04) and in the AKI group than in the acute on chronic renal failure (AKI-on-CRF) group (P <0.002). Tubular protein was higher in the AKI-on-CRF group (P <0.003) than in the AKI group. Tubular protein correlated with FENa in groups with diabetes mellitus (DM), AKI-on-CRF, and without hematuria (P <0.03, P <0.02 and P <0.004, respectively). Pattern of protein did not differ between groups with and without DM and clinical acute tubular necrosis (ATN). At the end of 40 months follow-up, category with predominantly glomerular protein progressed to chronic renal failure (CRF) or end-stage renal failure in higher proportion (P <0.05). In clinical AKI, we observed that glomerular protein dominated in cases with glomerular insult, as indicated by hematuria. Tubular protein was common in the study cases with CRF, DM and cases without hematuria. This indicates tubulo-interstitial injury for AKI in these cases. Patients with predominantly glomerular protein had an adverse outcome. PMID:21743220

  12. Detection of the end point temperature of thermal denatured protein in fish and chicken meat through SDS-PAGE electrophoresis

    NASA Astrophysics Data System (ADS)

    Gao, Hongwei; Mao, Mao; Liang, Chengzhu; Lin, Chao; Xiang, Jianhai

    2009-03-01

    Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied in the detection of the end point temperature (EPT) of thermal denatured protein in fish and meat in this study. It was also used in studying the thermal denatured temperature range of proteins in salmon and chicken meat. The results show that the temperature ranges of denatured proteins were from 65°C to 75°C, and these temperature ranges were influenced by the processing methods. Through SDS-PAGE, the features of repeated heating thermal denatured proteins under the same temperature and processing time were studied. The electrophoresis patterns of thermal denatured proteins determined through repeated heating at the same temperature did not exhibit any change. For the detection of cooked fish and meat samples, they were subjected to applying the SDS-PAGE method, which revealed an EPT ranging from 60°C to 80°C.

  13. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  14. Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle.

    PubMed

    Chaga, G; Hopp, J; Nelson, P

    1999-02-01

    A rapid method for the purification of lactate dehydrogenase from whole chicken muscle extract in one chromatographic step is reported. The purification procedure can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent is used that can be utilized at linear flow rates higher than 5 cm/min. The final preparation of the enzyme was with purity higher than 95% as ascertained by SDS-PAGE. Three immobilized metal ions (Ni2+, Zn2+ and Co2+) were compared for their binding properties towards the purified enzyme. The binding site of the enzyme for immobilized intermediate metal ions was determined after cleavage with CNBr and binding studies of the derivative peptides on immobilized Co2+. A peptide located on the N-terminus of the enzyme, implicated in the binding, has great potential as a purification tag in fusion proteins. PMID:9889081

  15. SDS-PAGE Analysis of Soluble Proteins in Reconstituted Milk Exposed to Different Heat Treatments

    PubMed Central

    Jovanovic, Snezana; Barac, Miroljub; Macej, Ognjen; Vucic, Tanja; Lacnjevac, Caslav

    2007-01-01

    This paper deals with the investigation of the impact of the heat treatment of reconstituted skim milk conducted at different temperatures, and the adding of demineralized whey on the protein solubility, soluble protein composition and interactions involved between proteins in a chemical complex. Commercial skim milk has been reconstituted and heat treated at 75°C, 85°C and 90°C for 20 minutes. Demineralized whey has been added in concentrations of 0.5%, 1.0 and 2.0%. The soluble protein composition has been determined by the polyacrilamide gel electrophoresis (SDS-PAGE) and by the densitometric analysis. Due to the different changes occurred during treatments at different temperatures, proteins of heat-treated samples containing added demineralized whey have had significantly different solubility. At lower temperatures (75°C and 85°C) the adding of demineralized whey decreased the protein solubility by 5.28%-26.41%, while the addition of demineralized whey performed at 90°C increased the soluble protein content by 5.61%-28.89%. Heat treatments, as well as the addition of demineralized whey, have induced high molecular weight complex formation. β-Lg, α-La and κ-casein are involved in high molecular weight complexes. The disulfide interactions between denatured molecules of these proteins are mostly responsible for the formation of coaggregates. The level of their interactions and the soluble protein composition are determined by the degree of temperature.

  16. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture. PMID:26584922

  17. Improving affinity chromatography resin efficiency using semi-continuous chromatography.

    PubMed

    Mahajan, Ekta; George, Anupa; Wolk, Bradley

    2012-03-01

    Protein A affinity chromatography is widely used for purification of monoclonal antibodies (MAbs) from harvested cell culture fluid (HCCF). At the manufacturing scale, the HCCF is typically loaded on a single Protein A affinity chromatography column in cycles until all of the HCCF is processed. Protein A resin costs are significant, comprising a substantial portion of the raw material costs in MAb manufacturing. Cost can be reduced by operating the process continuously using multiple smaller columns to a higher binding capacity in lieu of one industrial scale column. In this study, a series of experiments were performed using three 1-ml Hi-Trap™ MabSelect SuRe™ columns on a modified ÄKTA™ system operated according to the three Column Periodic Counter Current Chromatography (3C PCC) principle. The columns were loaded individually at different times until the 70% breakthrough point was achieved. The HCCF with unbound protein from the column was then loaded onto the next column to capture the MAb, preventing any protein loss. At any given point, all three columns were in operation, either loading or washing, enabling a reduction in processing time. The product yield and quality were evaluated and compared with a batch process to determine the effect of using the three column continuous process. The continuous operation shows the potential to reduce both resin volume and buffer consumption by ∼40%, however the system hardware and the process is more complex than the batch process. Alternative methods using a single standard affinity column, such as recycling load effluent back to the tank or increasing residence time, were also evaluated to improve Protein A resin efficiency. These alternative methods showed similar cost benefits but required longer processing time. PMID:22265178

  18. Isolation and identification of Enterococcus faecalis membrane proteins using membrane shaving, 1D SDS/PAGE, and mass spectrometry.

    PubMed

    Cathro, Peter; McCarthy, Peter; Hoffmann, Peter; Zilm, Peter

    2016-06-01

    Enterococcus faecalis is a significant nosocomial pathogen, which is able to survive in diverse environments and resist killing with antimicrobial therapies. The expression of cell membrane proteins play an important role in how bacteria respond to environmental stress. As such, the capacity to identify and study membrane protein expression is critical to our understanding of how specific proteins influence bacterial survival. Here, we describe a combined approach to identify membrane proteins of E. faecalis ATCC V583 using membranes fractionated by either 1D SDS/PAGE or membrane shaving, coupled with LC-ESI mass spectrometry. We identified 222 membrane-associated proteins, which represent approximately 24% of the predicted membrane-associated proteome: 170 were isolated using 1D SDS/PAGE and 68 with membrane shaving, with 36 proteins being common to both the techniques. Of the proteins identified by membrane shaving, 97% were membrane-associated with the majority being integral membrane proteins (89%). Most of the proteins identified with known physiology are involved with transportation across the membrane. The combined 1D SDS/PAGE and membrane shaving approach has produced the greatest number of membrane proteins identified from E. faecalis to date. These protocols will aid future researchers investigating changes in the membrane proteome of E. faecalis by improving our understanding of how E. faecalis adapts and responds to its environment. PMID:27419061

  19. SDS-PAGE and IR spectroscopy to evaluate modifications in the viral protein profile induced by a cationic porphyrinic photosensitizer.

    PubMed

    Costa, Liliana; Esteves, Ana Cristina; Correia, António; Moreirinha, Catarina; Delgadillo, Ivonne; Cunha, Ângela; Neves, Maria G P S; Faustino, Maria A F; Almeida, Adelaide

    2014-12-01

    Reactive oxygen species can be responsible for microbial photodynamic inactivation due to its toxic effects, which include severe damage to proteins, lipids and nucleic acids. In this study, the photo-oxidative modifications of the proteins of a non-enveloped T4-like bacteriophage, induced by the cationic porphyrin 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide were evaluated. Two methods were used: sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and infrared spectroscopy. SDS-PAGE analysis showed that the phage protein profile was considerably altered after photodynamic treatment. Seven protein bands putatively corresponding to capsid and tail tube proteins were attenuated and two other were enhanced. Infrared spectroscopy confirmed the time-dependent alteration on the phage protein profile detected by SDS-PAGE, indicative of a response to oxidative damage. Infrared analysis showed to be a promising and rapid screening approach for the analysis of the modifications induced on viral proteins by photosensitization. In fact, one single infrared spectrum can highlight the changes induced to all viral molecular structures, overcoming the delays and complex protocols of the conventional methods, in a much simple and cost effective way. PMID:25241141

  20. Exploring Fluorous Affinity by Liquid Chromatography.

    PubMed

    Catani, Martina; Guzzinati, Roberta; Marchetti, Nicola; Pasti, Luisa; Cavazzini, Alberto

    2015-07-01

    Terms such as "fluorous affinity" and "fluorophilicity" have been used to describe the unique partition and sorption properties often exhibited by highly fluorinated organic compounds, that is molecules rich in sp(3) carbon-fluorine bonds. In this work, we made use of a highly fluorinated stationary phase and a series of benzene derivatives to study the effect of one single perfluorinated carbon on the chromatographic behavior and adsorption properties of molecules. For this purpose, the adsorption equilibria of α,α,α-trifluorotoluene, toluene, and other alkylbenzenes have been studied by means of nonlinear chromatography in a variety of acetonitrile/water eluents. Our results reveal that one single perfluorinated carbon is already enough to induce a drastic change in the adsorption properties of molecules on the perfluorinated stationary phase. In particular, it has been found that adsorption is monolayer if the perfluoroalkyl carbon is present but that, when this unit is missing, molecules arrange as multilayer stack structures. These findings can contribute to the understanding of molecular mechanisms of fluorous affinity. PMID:26047527

  1. Frontal affinity chromatography (FAC): theory and basic aspects.

    PubMed

    Kasai, Ken-ichi

    2014-01-01

    Frontal affinity chromatography (FAC) is a versatile analytical tool for determining specific interactions between biomolecules and is particularly useful in the field of glycobiology. This article presents its basic aspects, merits, and theory. PMID:25117240

  2. Investigation of relationships between barley stress peptides and beer gushing using SDS-PAGE and MS screening.

    PubMed

    Hégrová, Blanka; Farková, Marta; Macuchová, Simona; Havel, Josef; Preisler, Jan

    2009-12-01

    The relationship between gushing and antifungal peptides in barley and malt kernels was examined for five barley varieties produced in the Czech Republic with four conditions of infection and treatment. Proteome changes during pathogen-seed interaction were observed with SDS-PAGE and MALDI-TOF MS. These methods were applied as a fast screening for observing the relationship between gushing and peptides/proteins. It was found that the presence of basic peptides, presumably hordothionins and non-specific lipid transfer protein type 1, did not correlate with the degree of gushing for malt (/r/ in <0.07, 0.34>), (/r/ in <0.01, 0.49>), respectively, as detected by both methods. PMID:19950350

  3. Isolation of the Binding Protein of Periplocoside E from BBMVs in Midgut of the Oriental Amyworm Mythimna separata Walker (Lepidoptera: Noctuidae) through Affinity Chromatography

    PubMed Central

    Feng, Mingxing; He, Zhenyu; Wang, Yuanyuan; Yan, Xiufang; Zhang, Jiwen; Hu, Zhaonong; Wu, Wenjun

    2016-01-01

    Periplocosides, which are insecticidal compounds isolated from the root bark of Periploca sepium Bunge, can affect the digestive system of insects. However, the mechanism though which periplocosides induces a series of symptoms remains unknown. In this study, affinity chromatography was conducted by coupling periplocoside E-semi-succinic acid ester with epoxy amino hexyl (EAH) sepharose 4B. Sodium dodecyl sulfonate-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed to analyze the fraction eluted by periplocoside E. Eight binding proteins (luciferin 4-monooxygenase, aminopeptidase N, aminopeptidase N3, nicotinamide adenine dinucleotide health (NADH) dehydrogenase subunit 5, phosphatidylinositol 3-phosphate 3-phosphatase myotubularin, actin, uncharacterized family 31 glucosidase KIAA1161, and 2OG-Fe(2) oxygenase superfamily protein) were obtained and identified through liquid chromatography/quadrupole-time of flight-mass spectrometry (LC/Q-TOF-MS) analysis of the midgut epithelium cells of Mythimna separata larvae. Aminopeptidase N and N3 are potential putative targets of periplocosides. This study establishes the foundation for further research on the mechanism of action and target localization of periplocosides in agricultural pests. PMID:27153092

  4. Isolation of the Binding Protein of Periplocoside E from BBMVs in Midgut of the Oriental Amyworm Mythimna separata Walker (Lepidoptera: Noctuidae) through Affinity Chromatography.

    PubMed

    Feng, Mingxing; He, Zhenyu; Wang, Yuanyuan; Yan, Xiufang; Zhang, Jiwen; Hu, Zhaonong; Wu, Wenjun

    2016-01-01

    Periplocosides, which are insecticidal compounds isolated from the root bark of Periploca sepium Bunge, can affect the digestive system of insects. However, the mechanism though which periplocosides induces a series of symptoms remains unknown. In this study, affinity chromatography was conducted by coupling periplocoside E-semi-succinic acid ester with epoxy amino hexyl (EAH) sepharose 4B. Sodium dodecyl sulfonate-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed to analyze the fraction eluted by periplocoside E. Eight binding proteins (luciferin 4-monooxygenase, aminopeptidase N, aminopeptidase N3, nicotinamide adenine dinucleotide health (NADH) dehydrogenase subunit 5, phosphatidylinositol 3-phosphate 3-phosphatase myotubularin, actin, uncharacterized family 31 glucosidase KIAA1161, and 2OG-Fe(2) oxygenase superfamily protein) were obtained and identified through liquid chromatography/quadrupole-time of flight-mass spectrometry (LC/Q-TOF-MS) analysis of the midgut epithelium cells of Mythimna separata larvae. Aminopeptidase N and N3 are potential putative targets of periplocosides. This study establishes the foundation for further research on the mechanism of action and target localization of periplocosides in agricultural pests. PMID:27153092

  5. Lab-on-a-chip and SDS-PAGE analysis of hemolymph protein profile from Rhipicephalus microplus (Acari: Ixodidae) infected with entomopathogenic nematode and fungus.

    PubMed

    Golo, Patrícia Silva; Dos Santos, Alessa Siqueira de Oliveira; Monteiro, Caio Marcio Oliveira; Perinotto, Wendell Marcelo de Souza; Quinelato, Simone; Camargo, Mariana Guedes; de Sá, Fillipe Araujo; Angelo, Isabele da Costa; Martins, Marta Fonseca; Prata, Marcia Cristina de Azevedo; Bittencourt, Vânia Rita Elias Pinheiro

    2016-09-01

    In the present study, lab-on-a-chip electrophoresis (LoaC) was suggested as an alternative method to the conventional polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) to analyze raw cell-free tick hemolymph. Rhipicephalus microplus females were exposed to the entomopathogenic fungus Metarhizium anisopliae senso latu IBCB 116 strain and/or to the entomopathogenic nematode Heterorhabditis indica LPP1 strain. Hemolymph from not exposed or exposed ticks was collected 16 and 24 h after exposure and analyze by SDS-PAGE or LoaC. SDS-PAGE yielded 15 bands and LoaC electrophoresis 17 bands. Despite the differences in the number of bands, when the hemolymph protein profiles of exposed or unexposed ticks were compared in the same method, no suppressing or additional bands were detected among the treatments regardless the method (i.e., SDS-PAGE or chip electrophoresis using the Protein 230 Kit®). The potential of LoaC electrophoresis to detect protein bands from tick hemolymph was considered more efficient in comparison to the detection obtained using the traditional SDS-PAGE method, especially when it comes to protein subunits heavier than 100 KDa. LoaC electrophoresis provided a very good reproducibility, and is much faster than the conventional SDS-PAGE method, which requires several hours for one analysis. Despite both methods can be used to analyze tick hemolymph composition, LoaC was considered more suitable for cell-free hemolymph protein separation and detection. LoaC hemolymph band percent data reported changes in key proteins (i.e., HeLp and vitellogenin) exceptionally important for tick embryogenesis. This study reported, for the first time, tick hemolymph protein profile using LoaC. PMID:27174026

  6. Prediction of Neutral Salt Elution Profiles for Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Stellwagen, Earle

    1981-04-01

    Neutral salts exhibit very marked differences as eluants of proteins from affinity columns. We observe: (i) that the relative potencies of neutral salts as eluants are independent of the protein or the affinity ligand in the systems studied, (ii) that the absolute salt concentration necessary to elute any given protein bound to the affinity matrix is proportional to the algebraic sum of a set of elution coefficients defined herein for the separate ions present in the solution, and (iii) that the proportionality between elution potency and elution coefficient is a function of the affinity of the protein for the immobilized ligand. Given the concentration of one neutral salt required for elution of a protein of interest from an affinity column, the elution capability of any neutral salt at any temperature can be quantitatively predicted for that protein. Accordingly, application and elution protocols for affinity chromatography can be designed to optimize the yield and fold purification of proteins.

  7. Exploiting unusual affinity of usual polysaccharides for separation of enzymes on fluidized beds.

    PubMed

    Roy; Sardar; Gupta

    2000-07-01

    Two polysaccharides, alginate and chitosan, showed unusual affinity and bound alpha-amylase (from various sources) and Aspergillus niger cellulase, respectively. The beads prepared from these polymers were successfully used for the purification of the respective enzymes by fluidized bed affinity chromatography. alpha-amylase from wheat germ could be purified by 58-fold with about 90% recovery of activity. Aspergillus niger cellulase, on the other hand, was purified by 30-fold with 80% recovery of enzyme activity. Both purified preparations show single band on SDS-PAGE. PMID:10862902

  8. Novel Cartilage Oligomeric Matrix Protein (COMP) Neoepitopes Identified in Synovial Fluids from Patients with Joint Diseases Using Affinity Chromatography and Mass Spectrometry*

    PubMed Central

    Åhrman, Emma; Lorenzo, Pilar; Holmgren, Kristin; Grodzinsky, Alan J.; Dahlberg, Leif E.; Saxne, Tore; Heinegård, Dick; Önnerfjord, Patrik

    2014-01-01

    To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDS-PAGE followed by in-gel digestion and mass spectrometric identification and characterization. Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided. PMID:24917676

  9. A surprising effect of extraction conditions on the mobility of some plant virus coat proteins in SDS-PAGE.

    PubMed

    Hassan, H; Borkhardt, B; Albrechtsen, M

    1994-02-01

    The coat protein from purified particles of pea seedborne mosaic potyvirus (PSbMV) moves in SDS-PAGE with an apparent molecular weight (M(r)) of 36 kDa. However, extracts of PSbMV infected plants prepared with SDS or urea contain PSbMV immunoreactive proteins with apparent M(r) 39 kDa as well as 36 kDa. The low mobility form may be generated from the apparent M(r) 36 kDa form by incubating purified PSbMV particles with healthy plant sap in the presence of denaturing agents. A similar effect is observed with bean yellow mosaic potyvirus, but not with three viruses outside the potyvirus group. Experiments suggest that a soluble plant enzyme is responsible for the conversion, which apparently takes place only in vitro under denaturing conditions. This phenomenon may lead to erroneous conclusions about the M(r) of some viral coat proteins. However, the conversion can be prevented by heat treatment of the plant tissue prior to extraction. PMID:8188819

  10. SDS-PAGE/immunoblot detection of Abeta multimers in human cortical tissue homogenates using antigen-epitope retrieval.

    PubMed

    Rosen, Rebecca F; Tomidokoro, Yasushi; Ghiso, Jorge A; Walker, Lary C

    2010-01-01

    The anomalous folding and polymerization of the beta-amyloid (Abeta) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis(1). Abeta is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into beta-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble Abeta multimers are more toxic than fibrillar Abeta amyloid(2). The identification and quantification of low- and high-molecular weight multimeric Abeta species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies(3). Naturally occurring Abeta multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with Abeta-specific antibodies. However, the separation and detection of multimeric Abeta requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the Abeta peptide. Using this protocol, we consistently detect Abeta monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology. PMID:20418805

  11. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transport. Partial purification of the nitrobenzylthioinosine-binding protein of pig erythrocytes by affinity chromatography.

    PubMed Central

    Agbanyo, F R; Vijayalakshmi, D; Craik, J D; Gati, W P; McAdam, D P; Asakura, J; Robins, M J; Paterson, A R; Cass, C E

    1990-01-01

    Derivatives of N6-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked derivatives of N6-(4-nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl beta-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 11C4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AG10 was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1% SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG10, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups. Images Fig. 5. Fig. 6. Fig. 7. PMID:2241896

  12. Kinetic Studies of Biological Interactions By Affinity Chromatography

    PubMed Central

    Schiel, John E.; Hage, David S.

    2009-01-01

    The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This review will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this review and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. PMID:19391173

  13. Specific capture of uranyl protein targets by metal affinity chromatography.

    PubMed

    Basset, Christian; Dedieu, Alain; Guérin, Philippe; Quéméneur, Eric; Meyer, Daniel; Vidaud, Claude

    2008-03-28

    To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO2(2+)) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of aminophosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis. PMID:18308325

  14. Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography.

    PubMed

    Li, Shangyong; Wang, Linna; Yang, Juan; Bao, Jing; Liu, Junzhong; Lin, Shengxiang; Hao, Jianhua; Sun, Mi

    2016-06-01

    In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. PMID:27058973

  15. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  16. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

  17. SDS-PAGE analysis of Aβ oligomers is disserving research into Alzheimer´s disease: appealing for ESI-IM-MS

    NASA Astrophysics Data System (ADS)

    Pujol-Pina, Rosa; Vilaprinyó-Pascual, Sílvia; Mazzucato, Roberta; Arcella, Annalisa; Vilaseca, Marta; Orozco, Modesto; Carulla, Natàlia

    2015-10-01

    The characterization of amyloid-beta peptide (Aβ) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aβ40 and Aβ42 oligomers of well-defined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aβ42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aβ oligomer samples.

  18. SDS-PAGE analysis of Aβ oligomers is disserving research into Alzheimer´s disease: appealing for ESI-IM-MS.

    PubMed

    Pujol-Pina, Rosa; Vilaprinyó-Pascual, Sílvia; Mazzucato, Roberta; Arcella, Annalisa; Vilaseca, Marta; Orozco, Modesto; Carulla, Natàlia

    2015-01-01

    The characterization of amyloid-beta peptide (Aβ) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aβ40 and Aβ42 oligomers of well-defined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aβ42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aβ oligomer samples. PMID:26450154

  19. SDS-PAGE analysis of Aβ oligomers is disserving research into Alzheimer´s disease: appealing for ESI-IM-MS

    PubMed Central

    Pujol-Pina, Rosa; Vilaprinyó-Pascual, Sílvia; Mazzucato, Roberta; Arcella, Annalisa; Vilaseca, Marta; Orozco, Modesto; Carulla, Natàlia

    2015-01-01

    The characterization of amyloid-beta peptide (Aβ) oligomer forms and structures is crucial to the advancement in the field of Alzheimer´s disease (AD). Here we report a critical evaluation of two methods used for this purpose, namely sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), extensively used in the field, and ion mobility coupled to electrospray ionization mass spectrometry (ESI-IM-MS), an emerging technique with great potential for oligomer characterization. To evaluate their performance, we first obtained pure cross-linked Aβ40 and Aβ42 oligomers of well-defined order. Analysis of these samples by SDS-PAGE revealed that SDS affects the oligomerization state of Aβ42 oligomers, thus providing flawed information on their order and distribution. In contrast, ESI-IM-MS provided accurate information, while also reported on the chemical nature and on the structure of the oligomers. Our findings have important implications as they challenge scientific paradigms in the AD field built upon SDS-PAGE characterization of Aβ oligomer samples. PMID:26450154

  20. Two-dimensional blue native/SDS-PAGE analysis of whole cell lysate protein complexes of rice in response to salt stress.

    PubMed

    Hashemi, Amenehsadat; Gharechahi, Javad; Nematzadeh, Ghorbanali; Shekari, Faezeh; Hosseini, Seyed Abdollah; Salekdeh, Ghasem Hosseini

    2016-08-01

    To understand the biology of a plant in response to stress, insight into protein-protein interactions, which almost define cell behavior, is thought to be crucial. Here, we provide a comparative complexomics analysis of leaf whole cell lysate of two rice genotypes with contrasting responses to salt using two-dimensional blue native/SDS-PAGE (2D-BN/SDS-PAGE). We aimed to identify changes in subunit composition and stoichiometry of protein complexes elicited by salt. Using mild detergent for protein complex solubilization, we were able to identify 9 protein assemblies as hetero-oligomeric and 30 as homo-oligomeric complexes. A total of 20 proteins were identified as monomers in the 2D-BN/SDS-PAGE gels. In addition to identifying known protein complexes that confirm the technical validity of our analysis, we were also able to discover novel protein-protein interactions. Interestingly, an interaction was detected for glycolytic enzymes enolase (ENO1) and triosephosphate isomerase (TPI) and also for a chlorophyll a-b binding protein and RuBisCo small subunit. To show changes in subunit composition and stoichiometry of protein assemblies during salt stress, the differential abundance of interacting proteins was compared between salt-treated and control plants. A detailed exploration of some of the protein complexes provided novel insight into the function, composition, stoichiometry and dynamics of known and previously uncharacterized protein complexes in response to salt stress. PMID:27362847

  1. [PHEMA/PEI]-Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma.

    PubMed

    Bakhshpour, Monireh; Derazshamshir, Ali; Bereli, Nilay; Elkak, Assem; Denizli, Adil

    2016-04-01

    The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]-Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]-Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). PMID:26838913

  2. [Progresses in screening active compounds from herbal medicine by affinity chromatography].

    PubMed

    Feng, Ying-shu; Tong, Shan-shan; Xu, Xi-ming; Yu, Jiang-nan

    2015-03-01

    Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening. PMID:26226740

  3. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  4. Molecular modeling of the affinity chromatography of monoclonal antibodies.

    PubMed

    Paloni, Matteo; Cavallotti, Carlo

    2015-01-01

    Molecular modeling is a methodology that offers the possibility of studying complex systems such as protein-ligand complexes from an atomistic point of view, making available information that can be difficultly obtained from experimental studies. Here, a protocol for the construction of molecular models of the interaction between antibodies and ligands that can be used for an affinity chromatography process is presented. The outlined methodology focuses mostly on the description of a procedure that may be adopted to determine the structure and free energy of interaction between the antibody and the affinity ligand. A procedure to extend the proposed methodology to include the effect of the environment (buffer solution, spacer, support matrix) is also briefly outlined. PMID:25749965

  5. Kinetic analysis of drug-protein interactions by affinity chromatography.

    PubMed

    Bi, Cong; Beeram, Sandya; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-10-01

    Information on the kinetics of drug-protein interactions is of crucial importance in drug discovery and development. Several methods based on affinity chromatography have been developed in recent years to examine the association and dissociation rates of these processes. These techniques include band-broadening measurements, the peak decay method, peak fitting methods, the split-peak method, and free fraction analysis. This review will examine the general principles and applications of these approaches and discuss their use in the characterization, screening and analysis of drug-protein interactions in the body. PMID:26724332

  6. Enrichment of Phosphopeptides via Immobilized Metal Affinity Chromatography.

    PubMed

    Swaney, Danielle L; Villén, Judit

    2016-03-01

    Immobilized metal affinity chromatography (IMAC) is a frequently used method for the enrichment of phosphorylated peptides from complex, cellular lysate-derived peptide mixtures. Here we outline an IMAC protocol that uses iron-chelated magnetic beads to selectively isolate phosphorylated peptides for mass spectrometry-based proteomic analysis. Under acidic conditions, negatively charged phosphoryl modifications preferentially bind to positively charged metal ions (e.g., Fe(3+), Ga(3+)) on the beads. After washing away nonphosphorylated peptides, a pH shift to basic conditions causes the elution of bound phosphopeptides from the metal ion. Under optimal conditions, very high specificity for phosphopeptides can be achieved. PMID:26933247

  7. Isolation of human lactate dehydrogenase isoenzyme X by affinity chromatography.

    PubMed Central

    Kolk, A H; van Kuyk, L; Boettcher, B

    1978-01-01

    Human isoenzyme LDH-X (lactate dehydrogenase isoenzyme X) was isolated from seminal fluid of frozen semen samples by affinity chromatography by using oxamate-Sepharose and AMP-Sepharose. In the presence of 1.6 mM-NAD+, isoenzyme LDH-X does not bind to AMP-Sepharose, whereas the other lactate dehydrogenase isoenzymes do. This is the crucial point in the isolation of isoenzyme LDH-X from the other isoenzymes. The purified human isoenzyme LDH-X had a specific activity of 146 units/mg of protein. Images Fig. 2. Fig. 3. PMID:213050

  8. Use of unsupervised and supervised artificial neural networks for the identification of lactic acid bacteria on the basis of SDS-PAGE patterns of whole cell proteins.

    PubMed

    Piraino, P; Ricciardi, A; Salzano, G; Zotta, T; Parente, E

    2006-08-01

    Conventional multivariate statistical techniques (hierarchical cluster analysis, linear discriminant analysis) and unsupervised (Kohonen Self Organizing Map) and supervised (Bayesian network) artificial neural networks were compared for as tools for the classification and identification of 352 SDS-PAGE patterns of whole cell proteins of lactic acid bacteria belonging to 22 species of the genera Lactobacillus, Leuconostoc, Enterococcus, Lactococcus and Streptococcus including 47 reference strains. Electrophoretic data were pre-treated using the logistic weighting function described by Piraino et al. [Piraino, P., Ricciardi, A., Lanorte, M. T., Malkhazova, I., Parente, E., 2002. A new procedure for data reduction in electrophoretic fingerprints of whole-cell proteins. Biotechnol. Lett. 24, 1477-1482]. Hierarchical cluster analysis provided a satisfactory classification of the patterns but was unable to discriminate some species (Leuconostoc, Lb. sakei/Lb. curvatus, Lb. acidophilus/Lb. helveticus, Lb. plantarum/Lb. paraplantarum, Lc. lactis/Lc. raffinolactis). A 7x7 Kohonen self-organizing map (KSOM), trained with the patterns of the reference strains, provided a satisfactory classification of the patterns and was able to discriminate more species than hierarchical cluster analysis. The map was used in predictive mode to identify unknown strains and provided results which in 85.5% of cases matched the classification obtained by hierarchical cluster analysis. Two supervised tools, linear discriminant analysis and a 23:5:2 Bayesian network were proven to be highly effective in the discrimination of SDS-PAGE patterns of Lc. lactis from those of other species. We conclude that data reduction by logistic weighting coupled to traditional multivariate statistical analysis or artificial neural networks provide an effective tool for the classification and identification of lactic acid bacteria on the basis of SDS-PAGE patterns of whole cell proteins. PMID:16480784

  9. Polymerization of SDS-PAGE gel by gamma irradiation and its use for characterization by electrophoresis of a protein [rapid communication

    NASA Astrophysics Data System (ADS)

    Kim, Dong-Ho; Kim, Jin-Hee; Seo, Ji-Hyun; Lee, Ju-Woon; Lim, Sang-Yong; Lee, Ho-Joon; Byun, Myung-Woo

    2005-12-01

    An SDS-PAGE gel strip was polymerized using a gamma irradiation process and used for electrophoresis. The relative mobility (Rf) and resolution of marker proteins were determined. Polymerization was induced by gamma irradiation in an acrylamide and N'-methylene bisacrylamide mixture with and without the polymerization initiators, ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED). The calibration curves of log 10 molecular weight of the protein versus the distance of the migration showed higher correlations in the gamma irradiated gel than in that of the APS-TEMED polymerized control. The Rf value of the protein was increased in the gel polymerized by gamma-irradiation.

  10. Purification of muscarinic acetylcholine receptors by affinity chromatography.

    PubMed Central

    André, C; De Backer, J P; Guillet, J C; Vanderheyden, P; Vauquelin, G; Strosberg, A D

    1983-01-01

    Calf forebrain homogenates contain 2.8 pM muscarinic acetylcholine receptors per mg of protein. [3H]Antagonist saturation binding experiments under equilibrium conditions revealed a single class of sites with equilibrium dissociation constants of 0.82 nM for [3H]dexetimide and 0.095 nM for [3H]quinuclidinyl benzilate. Displacement binding studies with agonists revealed the presence of low and high affinity sites. Here we describe the solubilization of muscarinic acetylcholine receptors with digitonin and their purification by affinity chromatography using an affinity gel which consisted of dexetimide coupled to Affi-Gel 10 (i.e., carboxy N-hydroxysuccinimide esters linked via a 1 nm spacer arm to agarose beads). Purified proteins were obtained by specific elution with muscarinic drugs, i.e., the antagonist atropine and the irreversible ligand propylbenzilylcholine mustard. SDS-polyacrylamide gel electrophoresis of the radioiodinated purified preparations revealed a major 70-K protein. Images Fig. 3. PMID:6605245

  11. Use of protein-protein interactions in affinity chromatography.

    PubMed

    Muronetz, V I; Sholukh, M; Korpela, T

    2001-10-30

    Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins. PMID:11694271

  12. Comparison of Inlet Geometry in Microfluidic Cell Affinity Chromatography

    PubMed Central

    Li, Peng; Tian, Yu; Pappas, Dimitri

    2011-01-01

    Cell separation based on microfluidic affinity chromatography is a widely used methodology in cell analysis research when rapid separations with high purity are needed. Several successful examples have been reported with high separation efficiency and purity; however, cell capture at the inlet area and inlet design has not been extensively described or studied. The most common inlets—used to connect the microfluidic chip to pumps, tubing, etc—are vertical (top-loading) inlets and parallel (in-line) inlets. In this work, we investigated the cell capture behavior near the affinity chip inlet area and compared the different performance of vertical inlet devices and parallel inlet devices. Vertical inlet devices showed significant cell capture capability near the inlet area, which led to the formation of cell blockages as the separation progressed. Cell density near the inlet area was much higher than the remaining channel, while for parallel inlet chips cell density at the inlet area was similar to the rest of the channel. In this paper, we discuss the effects of inlet type on chip fabrication, nonspecific binding, cell capture efficiency, and separation purity. We also discuss the possibility of using vertical inlets in negative selection separations. Our findings show that inlet design is critical and must be considered when fabricating cell affinity microfluidic devices. PMID:21207967

  13. Protein purification by aminosquarylium cyanine dye-affinity chromatography.

    PubMed

    Graça, Vânia C; Sousa, Fani; Santos, Paulo F; Almeida, Paulo S

    2015-01-01

    Affinity chromatography (AC) is one of the most important techniques for the separation and purification of biomolecules, being probably the most selective technique for protein purification. It is based on unique specific reversible interactions between the target molecule and a ligand. In this affinity interaction, the choice of the ligand is extremely important for the success of the purification protocol. The growing interest in AC has motivated an intense research effort toward the development of materials able to overcome the disadvantages of conventional natural ligands, namely their high cost and chemical and biological lability. In this context, synthetic dyes have emerged, in recent decades, as a promising alternative to biological ligands. Herein, detailed protocols for the assembling of a new chromatographic dye-ligand affinity support bearing an immobilized aminosquarylium cyanine dye on an agarose-based matrix (Sepharose CL-6B) and for the separation of a mixture o f three standard proteins: lysozyme, α-chymotrypsin, and trypsin are provided. PMID:25749942

  14. One step affinity recovery of 3α-hydroxysteroid dehydrogenase from cloned Escherichia coli.

    PubMed

    Yang, Hailin; Fang, Yanan; Wang, Zhizhen; Zhang, Ling

    2015-06-01

    3α-Hydroxysteroid dehydrogenase (3α-HSD), from Comamonas Testosterone, catalyze reversibly the oxidoreduction of 3α-hydroxyl groups of the steroid hormones. The gene encoding 3α-HSD (hsdA) from Comamonas Testosterone was expressed in Escherchia coli BL21 (DE3). A protocol for recovering 3α-HSD based on affinity strategy was designed and employed. Deoxycholic acid was chosen as the affinity ligand, and it was linked to Sepharose 4B with the aid of the spacers as cyanuric chloride and ethanediamine. With this specific affinity medium, the enzyme recovery process consisted of only one chromatography step to capture 3α-HSD. The target protein, analyzed on HPLC Agilent SEC-5 column, was of 94% pure among the captured protein, and 98% with SDS-PAGE analysis. The yield of the expressed enzyme was 8.8% of crude extracted proteins; the recovery yield of 3α-HSD was 73.2%. 3α-HSD was revealed as a non-covalent homodimer with molecular mass of ∼56kDa by 15.0% SDS-PAGE analysis and SE-HPLC analysis. The desorption constant Kd and the theoretical maximum absorption Qmax on the affinity medium were 4.5μg/g medium and 21.3mg/g medium, respectively. PMID:25913427

  15. Determination of the bovine food allergen casein in white wines by quantitative indirect ELISA, SDS-PAGE, Western blot and immunostaining.

    PubMed

    Patrick, Weber; Hans, Steinhart; Angelika, Paschke

    2009-09-23

    This study describes the characterization of allergic bovine casein and caseinate fining agents by SDS-PAGE analysis and the development of a quantitative indirect ELISA for the detection of these substances in wines. The ELISA was applied to various experimental wines that were treated with different caseinate dosages and went through different processing steps and to a panel of commercial wines. Positive results were assured by SDS-PAGE, Western blot, and immunostaining. Comprehensive literature research was done to evaluate the demanded sensitivity of the ELISA. The results showed that alpha- and beta-caseins remain in some wines and are detectable. Estimated amounts were in the range or below an estimated no-observed adverse effect level (NOAEL) of 0.9 mg/L, but it was concluded that there is still an uncertainty about this NOAEL. Additional applied processing, referring to bentonite treatment and successive filtration, was determined to contribute to a significant decrease of casein residues in wines. PMID:19754170

  16. Comparative analysis of the human urinary proteome by 1D SDS-PAGE and chip-HPLC-MS/MS identification of the AACT putative urinary biomarker.

    PubMed

    Zhang, Yuan; Li, Yanyan; Qiu, Feng; Qiu, Zongyin

    2010-12-15

    Urine is one of the most attractive analyte used for clinical diagnosis. NSCLC (non-small cell lung carcinoma), which includes adenocarcinoma, squamous cell carcinoma and large-cell carcinoma, is a leading cause of cancer-related deaths. In the present study, urinary proteomes of normal individuals and NSCLC patients were compared using 1D SDS-PAGE. From the distinctly differentially expressed bands in SDS-PAGE gel, 40 proteins were identified by chip-HPLC-MS/MS, including five proteins relevant to NSCLC. One of the selected proteins, alpha-1-antichymotrypsin (AACT), was further validated in urine by western blot and in lung tissue by immunohistochemistry staining. Higher expression level of AACT in NSCLC patients was observed by western blot when compared with normal urine samples. Significantly, the NSCLC tumor tissue (18 out of 20 cases, 90%) showed a significantly higher expression level of AACT compared to adjacent non-tumor lung tissue (3 out of 20 cases, 15%). These results establish AACT as a potential biomarker for objective and non-invasive diagnosis of NSCLC in urine and the other four NSCLC-related proteins were also listed. PMID:21093387

  17. Search for Amyloid-Binding Proteins by Affinity Chromatography

    PubMed Central

    Calero, Miguel; Rostagno, Agueda; Ghiso, Jorge

    2013-01-01

    ‘Amyloid binging proteins’ is a generic term used to designate proteins that interact with different forms of amyloidogenic peptides or proteins and that, as a result, may modulate their physiological and pathological functions by altering solubility, transport, clearance, degradation, and fibril formation. We describe a simple affinity chromatography protocol to isolate and characterize amyloid-binding proteins based on the use of sequential elution steps that may provide further information on the type of binding interaction. As an example, we depict the application of this protocol to the study of Alzheimer’s amyloid β (Aβ) peptide-binding proteins derived from human plasma. Biochemical analysis of the proteins eluted under different conditions identified serum amyloid P component (SAP) and apolipoprotein J (clusterin) as the main plasma Aβ-binding proteins while various apolipoproteins (apoA-IV, apoE, and apoA-I), as well as albumin (HSA) and fibulin were identified as minor contributors. PMID:22528093

  18. Evidence for monomeric and oligomeric hormone-binding domains in affinity-purified gonadotropin receptor from rat ovary

    SciTech Connect

    Zhang, Q.Y.; Menon, K.M.J. )

    1989-11-01

    Rat ovarian lutropin/choriogonadotropin receptor was purified from a Triton X-100-solubilized membrane preparation by affinity chromatography with Affi-Gel 10 coupled to purified human choriogonadotropin. The affinity-purified receptor preparations contained a single class of high-affinity binding sites for {sup 125}I-labeled human choriogonadotropin, with an equilibrium dissociation constant (K{sub d}) of 2.5 {times} 10{sup {minus}9} M, which is comparable to the K{sub d} values for membrane-bound and solubilized receptors. The purified receptor appeared as two dominant bands with molecular weights of 135,000 and 92,000 after sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) under nonreducing conditions. When the individual affinity-purified receptor bands were electroeluted from the gel and analyzed again by SDS/PAGE under nonreducing conditions, both the M{sub r} 92,000 and the 135,000 proteins retained their original molecular form even when 8 M urea was included in the gel. However, when the electrophoretically purified M{sub r} 92,000 and 135,000 bands were subjected to SDS/PAGE under reducing conditions, the M{sub r} 135,000 species was almost completely converted to a M{sub r} 92,000 band, but the M{sub r} 92,000 species did not undergo any alteration in molecular weight. The results suggest that the lutropin/choriogonadotropin receptor from rat ovary exists in two molecular forms, and the higher molecular weight form appears to be composed of disulfide-linked M{sup r} 92,000 subunit, which comprises the hormone-binding domain.

  19. Comparison of low molecular weight glutenin subunits identified by SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR in common wheat

    PubMed Central

    2010-01-01

    Background Low-molecular-weight glutenin subunits (LMW-GS) play a crucial role in determining end-use quality of common wheat by influencing the viscoelastic properties of dough. Four different methods - sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE, IEF × SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR), were used to characterize the LMW-GS composition in 103 cultivars from 12 countries. Results At the Glu-A3 locus, all seven alleles could be reliably identified by 2-DE and PCR. However, the alleles Glu-A3e and Glu-A3d could not be routinely distinguished from Glu-A3f and Glu-A3g, respectively, based on SDS-PAGE, and the allele Glu-A3a could not be differentiated from Glu-A3c by MALDI-TOF-MS. At the Glu-B3 locus, alleles Glu-B3a, Glu-B3b, Glu-B3c, Glu-B3g, Glu-B3h and Glu-B3j could be clearly identified by all four methods, whereas Glu-B3ab, Glu-B3ac, Glu-B3ad could only be identified by the 2-DE method. At the Glu-D3 locus, allelic identification was problematic for the electrophoresis based methods and PCR. MALDI-TOF-MS has the potential to reliably identify the Glu-D3 alleles. Conclusions PCR is the simplest, most accurate, lowest cost, and therefore recommended method for identification of Glu-A3 and Glu-B3 alleles in breeding programs. A combination of methods was required to identify certain alleles, and would be especially useful when characterizing new alleles. A standard set of 30 cultivars for use in future studies was chosen to represent all LMW-GS allelic variants in the collection. Among them, Chinese Spring, Opata 85, Seri 82 and Pavon 76 were recommended as a core set for use in SDS-PAGE gels. Glu-D3c and Glu-D3e are the same allele. Two new alleles, namely, Glu-D3m in cultivar Darius, and Glu-D3n in Fengmai 27, were identified by 2-DE. Utilization of the suggested standard cultivar set, seed of

  20. Genetic variation of jointed goatgrass (Aegilops cylindrica Host.) from Iran using RAPD-PCR and SDS-PAGE of seed proteins.

    PubMed

    Farkhari, M; Naghavi, M R; Pyghambari, S A; Sabokdast

    2007-09-01

    Genetic variation of 28 populations of jointed goatgrass (Aegilops cylindrica Host.), collected from different parts of Iran, were evaluated using both RAPD-PCR and SDS-PAGE of seed proteins. The diversity within and between populations for the three-band High Molecular Weight (HMW) subunits of glutenin pattern were extremely low. Out of 15 screened primers of RAPD, 14 primers generated 133 reproducible fragments which among them 92 fragments were polymorphic (69%). Genetic similarity calculated from the RAPD data ranged from 0.64 to 0.98. A dendrogram was prepared on the basis of a similarity matrix using the UPGMA algorithm and separated the 28 populations into two groups. Confusion can happen between populations with the same origin as well as between populations of very diverse geographical origins. Our results show that compare to seed storage protein, RAPD is suitable for genetic diversity assessment in Ae. cylindrica populations. PMID:19090190

  1. Validation of Cis and Trans Modes in Multistep Phosphotransfer Signaling of Bacterial Tripartite Sensor Kinases by Using Phos-Tag SDS-PAGE

    PubMed Central

    Kinoshita-Kikuta, Emiko; Kinoshita, Eiji; Eguchi, Yoko; Koike, Tohru

    2016-01-01

    Tripartite sensor kinases (TSKs) have three phosphorylation sites on His, Asp, and His residues, which are conserved in a histidine kinase (HK) domain, a receiver domain, and a histidine-containing phosphotransmitter (HPt) domain, respectively. By means of a three-step phosphorelay, TSKs convey a phosphoryl group from the γ-phosphate group of ATP to the first His residue in the HK domain, then to the Asp residue in the receiver domain, and finally to the second His residue in the HPt domain. Although TSKs generally form homodimers, it was unknown whether the mode of phosphorylation in each step was intramolecular (cis) or intermolecular (trans). To examine this mode, we performed in vitro complementation analyses using Ala-substituted mutants of the ATP-binding region and three phosphorylation sites of recombinant ArcB, EvgS, and BarA TSKs derived from Escherichia coli. Phosphorylation profiles of these kinases, determined by using Phos-tag SDS-PAGE, showed that the sequential modes of the three-step phosphoryl-transfer reactions of ArcB, EvgS, and BarA are all different: cis-trans-trans, cis-cis-cis, and trans-trans-trans, respectively. The inclusion of a trans mode is consistent with the need to form a homodimer; the fact that all the steps for EvgS have cis modes is particularly interesting. Phos-tag SDS-PAGE therefore provides a simple method for identifying the unique and specific phosphotransfer mode for a given kinase, without taking complicated intracellular elements into consideration. PMID:26828204

  2. Comparative examination of adsorption of serum proteins on HSA- and PLGA-based nanoparticles using SDS-PAGE and LC-MS.

    PubMed

    Gossmann, R; Fahrländer, E; Hummel, M; Mulac, D; Brockmeyer, J; Langer, K

    2015-06-01

    The behavior of nanosized drug carrier systems under cell culture conditions and therefore also the destiny in the body are highly influenced by the protein corona, which is formed upon entering a biological environment. Some of the adsorbed proteins, named opsonins, lead to a shortened plasma circulation half-life of the nanoparticles. Others are attributed to promote the transport of nanoparticles into other compartments of the body, just to mention two examples. Hence, detailed knowledge concerning the composition of the protein corona is of great importance. The aim of this work was to investigate the influence of the nanoparticle starting material and the surface modification on the composition of the adsorbed serum proteins in a cell culture environment. Therefore, positively charged nanoparticles based on the biodegradable polymer poly(dl-lactide-co-glycolide) (PLGA) stabilized with didodecyldimethylammonium bromide (DMAB) and negatively charged nanoparticles based on human serum albumin (HSA) were prepared and modified with hydrophilic polymers. By incubating the nanoparticles with fetal bovine serum (FBS) the adsorption of serum proteins on the colloidal system was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) a semi-quantitative analysis of the protein corona was performed and after enzymatic in-solution-digestion the adsorbed proteins were identified using high resolution LC-MS. Our study accentuates the influence of the core material, surface charge, and surface modification on the amount and nature of the adsorbed proteins. The combination of SDS-PAGE and LC-MS turns out to be a simple and reliable method to investigate the protein corona of nanoparticles. PMID:25813886

  3. Studying the Stability of S-Layer Protein of Lactobacillus Acidophilus ATCC 4356 in Simulated Gastrointestinal Fluids Using SDS-PAGE and Circular Dichroism

    PubMed Central

    Eslami, Neda; Kermanshahi, Rouha Kasra; Erfan, Mohammad

    2013-01-01

    Crystalline arrays of proteinaceous subunits forming surface layers (S-layers) are now recognized as one of the most common outermost cell envelope components of prokaryotic organisms. The surface layer protein of Lactobacillus acidophilus ATCC4356 is composed of a single species of protein of apparent molecular weight of 43-46 KDa. Considering the Lactobacillus acidophilus ATCC4356 having the S-layer is stable in harsh gastrointestinal (GI) conditions, a protective role against destructive GI factors which has been proposed for these nanostructures. It opens interesting perspectives in the using and development of this S-layer as a protective coat for oral administration of unstable drug nanocarriers. To achieve this goal, it is necessary to study the in-vitro stability of the S-layers in the simulated gastrointestinal fluids (SGIF). This study was planned to evaluate the in-vitro stability of the extracted S-layer protein of Lactobacillus acidophilus ATCC4356 in SGIF using it as a protective coat in oral drug delivery. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and circular dichroism (CD) spectroscopy were used to study the stability of the S-layer protein incubated in SGIF. Both the SDS-PAGE and CD spectra results showed that Lactobacillus acidophilus ATCC4356 S-layer protein is stable in simulated gastric fluid (SGF) with pH = 2 up to 5 min. It is stable in SGF pH = 3.2 and above it, with and without pepsin. It is also stable in all the simulated intestinal fluids. This S-layer is also stable in all of the simulated intestinal fluids. PMID:24250671

  4. Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

    SciTech Connect

    Weslake, R.J.; Chesney, S.L.; Petkau, A.; Friesen, A.D.

    1986-05-15

    Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.

  5. Mining the soluble chloroplast proteome by affinity chromatography.

    PubMed

    Bayer, Roman G; Stael, Simon; Csaszar, Edina; Teige, Markus

    2011-04-01

    Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO(2), they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions. PMID:21365755

  6. Mining the soluble chloroplast proteome by affinity chromatography

    PubMed Central

    Bayer, Roman G; Stael, Simon; Csaszar, Edina; Teige, Markus

    2011-01-01

    Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO2, they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions. PMID:21365755

  7. Polymer versus monomer as displacer in immobilized metal affinity chromatography.

    PubMed

    Arvidsson, P; Ivanov, A E; Galaev IYu; Mattiasson, B

    2001-04-01

    Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu2+-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48 degrees C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)6-tag [(His)6-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu2+ ion. Complete elution of bound (His)6-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)6-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature. PMID:11334341

  8. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    ERIC Educational Resources Information Center

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  9. Affinity Chromatography of Lactate Dehydrogenase: An Experiment for the Undergraduate Biochemistry Laboratory.

    ERIC Educational Resources Information Center

    Anderson, Alexander J.

    1988-01-01

    Discusses a laboratory technique of enzyme purification by affinity chromatography as part of an undergraduate biochemical methodology course. Provides preparation details of the rat muscle homogenate and reagents. Proposes column requirements and assaying information. (MVL)

  10. [Urine protein analysis with the sodium-dodecyl-sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) in healthy cats and cats with kidney diseases].

    PubMed

    Meyer-Lindenberg, A; Wohlsein, P; Trautwein, G; Nolte, I

    1997-03-01

    In this investigation, the value of urine protein analysis by means of molecular-weight related sodium dodecyl-polyacryl gradient gel electrophoresis (SDS-PAGE) was examined with regard to its applicability and diagnostic significance in nephropathy in the cat. A total of 87 cats was included in the study, 30 of them that were clinically healthy served as the control group. The urine protein pattern of this group had, besides the band representing the market albumin, and additional broad band within the size of the marker transferrin. In some cases, weak bands were present within the range of the Tamm-Horsfall-protein and immunoglobulin G. Micromolecular protein bands were not demonstrable. The remaining 57 animals had a histologically proven nephropathy. Thirty-eight cats had elevated urea and/or creatinine values in the plasma (group 1), and 19 animals had values within the reference range (group 2). The urine protein pattern as evidenced by SDS-urine electrophoresis was altered in all cats with histologically proven nephropathy, and it is thus concluded that with this technique a nephropathy can be diagnosed very early and prior to changes of plasma urea and creatinine (group 2). Moreover, in most of the cases, the nephrological changes can be classified as glomerular or tubulo-interstitial (group 1 and group 2). However, it is not possible to draw exact conclusions concerning the underlying morphological changes, nor can the severity of the disease be correctly assessed. PMID:9123982

  11. A Phos-tag SDS-PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1.

    PubMed

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Kubota, Yuji; Takekawa, Mutsuhiro; Koike, Tohru

    2016-07-01

    MEK1, an essential component of the mitogen-activated protein kinase (MAPK) pathway, is phosphorylated during activation of the pathway; 12 phosphorylation sites have been identified in human MEK1 by MS-based phosphoproteomic methods. By using Phos-tag SDS-PAGE, we found that multiple variants of MEK1 with different phosphorylation states are constitutively present in typical human cells. The Phos-tag-based strategy, which makes effective use of existing information on the location of phosphorylation sites, permits quantitative time-course profiling of MEK1 phosphospecies in their respective phosphorylation states. By subsequent immunoblotting with an anti-HaloTag antibody, we analyzed a HaloTag-fused MEK1 protein and 12 potential phosphorylation-site-directed mutants of the protein transiently expressed in HEK 293 cells. This strategy revealed that MEK1 is constitutively and mainly phosphorylated at the Thr-292, Ser-298, Thr-386, and Thr-388 residues in vivo, and that combinations of phosphorylations at these four residues produce at least six phosphorylated variants of MEK1. Like the levels of phosphorylation of the Ser-218 and Ser-222 residues by RAF1, which have been well studied, the phosphorylation statuses of Thr-292, Ser-298, Thr-386, and Thr-388 residues vary widely during activation and deactivation of the MAPK pathway. Furthermore, we demonstrated inhibitor-specific profiling of MEK1 phosphospecies by using three MEK inhibitors: TAK-733, PD98059, and U0126. PMID:27169363

  12. Frontal affinity chromatography: A unique research tool for biospecific interaction that promotes glycobiology

    PubMed Central

    KASAI, Kenichi

    2014-01-01

    Combination of bioaffinity and chromatography gave birth to affinity chromatography. A further combination with frontal analysis resulted in creation of frontal affinity chromatography (FAC). This new versatile research tool enabled detailed analysis of weak interactions that play essential roles in living systems, especially those between complex saccharides and saccharide-binding proteins. FAC now becomes the best method for the investigation of saccharide-binding proteins (lectins) from viewpoints of sensitivity, accuracy, and efficiency, and is contributing greatly to the development of glycobiology. It opened a door leading to deeper understanding of the significance of saccharide recognition in life. The theory is also concisely described. PMID:25169774

  13. Frontal affinity chromatography: a unique research tool for biospecific interaction that promotes glycobiology.

    PubMed

    Kasai, Kenichi

    2014-01-01

    Combination of bioaffinity and chromatography gave birth to affinity chromatography. A further combination with frontal analysis resulted in creation of frontal affinity chromatography (FAC). This new versatile research tool enabled detailed analysis of weak interactions that play essential roles in living systems, especially those between complex saccharides and saccharide-binding proteins. FAC now becomes the best method for the investigation of saccharide-binding proteins (lectins) from viewpoints of sensitivity, accuracy, and efficiency, and is contributing greatly to the development of glycobiology. It opened a door leading to deeper understanding of the significance of saccharide recognition in life. The theory is also concisely described. PMID:25169774

  14. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies.

    PubMed

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  15. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies

    PubMed Central

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  16. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  17. Purification of phage display-modified bacteriophage T4 by affinity chromatography

    PubMed Central

    2011-01-01

    Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be

  18. SDS-PAGE Analysis of the Outer Membrane Proteins of Uropathogenic Escherichia coli Isolated from Patients in Different Wards of Nemazee Hospital, Shiraz, Iran

    PubMed Central

    Dehghani, Behzad; Mottamedifar, Mohammad; Khoshkharam-Roodmajani, Hossein; Hassanzadeh, Amir; Zomorrodian, Kamyar; Rahimi, Amir

    2016-01-01

    Background: Outer membrane proteins (OMPs) constitute the main structure and about half of the cell wall of Gram-negative bacteria. The OMPs of Escherichia coli (E. coli) play an important role in its drug resistance. Previous studies have shown that the OMPs of E. coli enhance its pathogenic effects by helping the bacterium to evade the immune defense and promote its adsorption to host cells. We sought to compare E. coli isolates collected from different hospital wards and to perform a primary investigation of the association between the serotypes and profiles of their OMPs. We also aimed to detect the diversity of the E. coli isolates from the hospitalized patients. Methods: A total of 115 isolates of E. coli were collected from patients hospitalized in Nemazee Hospital, Shiraz, Iran. After biochemical and serological tests, OMPs were extracted by using glass beads and N-Lauroylsarcosine sodium. OMP typing was done by 10% SDS-PAGE and Coomassie brilliant blue staining. In terms of the number of protein bands, OMP-I was detected with 2 bands, OMP-α with 3 bands, and OMP-β with1 band. Results: Of the 115 isolates, 103 were OMP-I and 12 were OMP-α; none of the isolates belonged to OMP-β. Our statistical analyses showed a relationship between OMP patterns and other factors, including hospital wards and source of samples. Serotyping showed that the majority of the isolates were O128. Conclusion: Our results demonstrated some similarities between the OMP band patterns of the analyzed groups of E. coli. Of all the OMPs in the isolates from the hospitalized and outpatient department patients, OmpA and OmpC were the most prevalent proteins in the outer membrane of the studied uropathogenic E. coli. PMID:27582589

  19. Comparison the effect of three commercial enzymes for enzymatic hydrolysis of two substrates (rice bran protein concentrate and soy-been protein) with SDS-PAGE.

    PubMed

    Ahmadifard, Nasrollah; Murueta, Julio Humberto Cordova; Abedian-Kenari, Abdolmohammad; Motamedzadegan, Ali; Jamali, Hadi

    2016-02-01

    In this research enzymatic hydrolysis of rice bran protein concentrate (RBPC) and soybean Protein (SBP) as control were studied with 3 commercial enzymes (Alcalase®, Papain and acommercial 3-enzyme cocktail containing of 1.6 mg ml(-1) Trypsin, 3.1 mg ml(-1) Chymotrypsin, 1.3 mg ml(-1)Aminopeptidase (SIGMA P7500) and 7.95 mg ml(-1)pronase type XIV (SIGMA P5147) by the pH stat method. The hydrolysis was carried out at temperature of 28 C, 60 min and pH 8.00. Results were showed that RBPC, and SBP had higher Degree hydrolysis (DH %) with Alcalase® enzyme. Alcalase®had stronger capability for hydrolysis compared to the other tested enzymes. After 60 minute of hydrolysis time, the DH% of Alcalase® for RBPC and SBP was 12.69 and 12.50 %, respectively. In contrast, papain enzyme was showed lowest DH% in three substrates that 1.56 and 1.24 % were for SBP and RBPC, respectively.The hydrolysis of the protein fraction performed the three enzymes on the two substrates was followed in SDS-PAGE. RBPC and SBP showed almost complete digestion with Alcalase® enzyme after 60 minutes. 3-enzyme cocktail enzyme hydrolyzed better the RBPC than the SBP. Papain enzyme had less effect on the two substrates than other 2 enzymes. It was found that Alcalase® has highest capability for hydrolysis compared to other enzyme preparations. The high value protein hydrolysates prepared by Alcalase® can be used as value added ingredients in many food formulations. They are also suitable for a broad range of industrial food applications and also for cosmetic and personal care products. PMID:27162408

  20. Affinity chromatography based on a combinatorial strategy for rerythropoietin purification.

    PubMed

    Martínez-Ceron, María C; Marani, Mariela M; Taulés, Marta; Etcheverrigaray, Marina; Albericio, Fernando; Cascone, Osvaldo; Camperi, Silvia A

    2011-05-01

    Small peptides containing fewer than 10 amino acids are promising ligand candidates with which to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X = Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser, or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized, and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 μM were obtained. The best two peptides were immobilized on Sepharose, and the resultant chromatographic matrixes showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 μM. Chinese hamster ovary (CHO) cell culture supernatant was spiked with rhEPO, and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90% and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose, respectively. PMID:21495625

  1. Negative Enrichment of Target Cells by Microfluidic Affinity Chromatography

    PubMed Central

    Li, Peng; Gao, Yan; Pappas, Dimitri

    2011-01-01

    A three-dimensional microfluidic channel was developed for high purity cell separations. This system featured high capture affinity using multiple vertical inlets to an affinity surface. In cell separations, positive selection (capture of the target cell) is usually employed. Negative enrichment, the capture of non-target cells and elution of target cells, has distinct advantages over positive selection. In negative enrichment, target cells are not labeled, and are not subjected to strenuous elution conditions or dilution. As a result, negative enrichment systems are amenable to multi-step processes in microfluidic systems. In previous work, we reported cell capture enhancement effects at vertical inlets to the affinity surface. In this study, we designed a chip that has multiple vertical and horizontal channels, forming a three-dimensional separation system. Enrichment of target cells showed separation purities of 92-96%, compared with straight-channel systems (77% purity). A parallelized chip was also developed for increased sample throughput. A two-channel showed similar separation purity with twice the sample flow rate. This microfluidic system, featuring high separation purity, ease of fabrication and use, is suitable for cell separations when subsequent analysis of target cells is required. PMID:21939198

  2. Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

    PubMed

    Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

    2016-04-01

    Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods. PMID:26973166

  3. Purification of a Recombinant Polyhistidine-Tagged Glucosyltransferase Using Immobilized Metal-Affinity Chromatography (IMAC).

    PubMed

    de Costa, Fernanda; Barber, Carla J S; Pujara, Pareshkumar T; Reed, Darwin W; Covello, Patrick S

    2016-01-01

    Short peptide tags genetically fused to recombinant proteins have been widely used to facilitate detection or purification without the need to develop specific procedures. In general, an ideal affinity tag would allow the efficient purification of tagged proteins in high yield, without affecting its function. Here, we describe the purification steps to purify a recombinant polyhistidine-tagged glucosyltransferase from Centella asiatica using immobilized metal affinity chromatography. PMID:26843168

  4. Mixed-bed affinity chromatography: principles and methods.

    PubMed

    Boschetti, Egisto; Righetti, Pier Giorgio

    2015-01-01

    Mixed-bed chromatography is far from being a well-established technology within the panoply of bioseparation tools. Composed of an assembly of distinct sorbents that are mixed in a single bed, they have been mostly developed in the last decade for the reduction of dynamic concentration range where they allowed discovering many low-copy proteins within very complex proteomes. Other interesting preparative applications of mixed-bed chromatography have since been developed. In this chapter the basic concepts first and then detailed application recipes are described for (1) the reduction of protein dynamic concentration range, (2) the removal of impurity traces at the last stage of a biopurification process, and (3) the selection and use of sorbents as mixed bed in protein purification. PMID:25749952

  5. Dimerization Capacities of FGF2 Purified with or without Heparin-Affinity Chromatography

    PubMed Central

    Chiu, Liang-Yuan; Taouji, Said; Moroni, Elisabetta; Colombo, Giorgio; Chevet, Eric; Sue, Shih-Che; Bikfalvi, Andreas

    2014-01-01

    Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well. PMID:25299071

  6. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    PubMed

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario. PMID:25748537

  7. Membrane affinity chromatography used for the separation of trypsin inhibitor.

    PubMed

    Guo, W; Shang, Z; Yu, Y; Guan, Y; Zhou, L

    1992-01-01

    Polysulphone (PS) was chemically modified by acrylation-amination and by chloromethylation-amination, respectively. An ultrafiltration membrane of chemically modified polysulphone (CMPS) was prepared by the phase inversion method. Trypsin was then covalently bonded onto the CMPS membrane by diazotization. The activity of immobilized trypsin reaches up to 10200 U/g; 15 mg trypsin was immobilized on 1 g CMPS membrane. Separation of soybean trypsin inhibitor was carried out on the affinity membrane, yielding 6.5 mg pure trypsin inhibitor in one run. The enzyme membrane has good activity and stability. PMID:1638098

  8. Affinity monolith chromatography: A review of principles and recent analytical applications

    PubMed Central

    Pfaunmiller, Erika L.; Paulemond, Marie Laura; Dupper, Courtney M.; Hage, David S.

    2012-01-01

    Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis or studies of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments or applications of this method, with particular emphasis being given to work that has appeared in the last five years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal-ions and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing and biotechnology. Current trends and possible future directions in AMC are also discussed. PMID:23187827

  9. A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

    PubMed

    Dai, Lu; Li, Weikang; Sun, Fei; Li, Baizhi; Li, Hongrui; Zhang, Hongxing; Zheng, Qingchuan; Liang, Chongyang

    2016-09-01

    Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media. PMID:27524303

  10. Selective retention of basic compounds by metal aquo-ion affinity chromatography.

    PubMed

    Asakawa, Yoshiki; Yamamoto, Eiichi; Asakawa, Naoki

    2014-10-01

    A novel metal aquo-ion affinity chromatography has been developed for the analysis of basic compounds using heat-treated silica gel containing hydrated metal cations (metal aquo-ions) as the packing material. The packing materials of the metal aquo-ion affinity chromatography were prepared by the immobilization of a single metal component such as Fe(III), Al(III), Ag(I), and Ni(II) on silica gel followed by extensive heat treatment. The immobilized metals form aquo-ions to present cation-exchange ability for basic analytes and the cation-exchange ability for basic analytes depends on pKa of the immobilized metal species. In the present study, to evaluate the retention characteristics of metal aquo-ion affinity chromatography, the on-line solid-phase extraction of drugs was investigated. Obtained data clearly evidence the selective retention capability of metal aquo-ion affinity chromatography for basic analytes with sufficient capacity. PMID:25044622

  11. Glycan-specific whole cell affinity chromatography: a versatile microbial adhesion platform

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have constructed a C-glycoside ketohydrazide affinity chromatography resin that interacts with viable whole-cell microbial populations with biologically appropriate stereo-specificity in a carbohydrate-defined manner. It readily allows for the quantification, selection, and manipulation of target...

  12. The quest for affinity chromatography ligands: are the molecular libraries the right source?

    PubMed

    Perret, Gérald; Santambien, Patrick; Boschetti, Egisto

    2015-08-01

    Affinity chromatography separations of proteins call for highly specific ligands. Antibodies are the most obvious approach; however, except for specific situations, technical and economic reasons are arguments against this choice especially for preparative purposes. With this in mind, the rationale is to select the most appropriate ligands from collections of pre-established molecules. To reach the objective of having a large structural coverage, combinatorial libraries have been proposed. These are classified according to their nature and origin. This review presents and discusses the most common affinity ligand libraries along with the most appropriate screening methods for the identification of the right affinity chromatography selective structure according to the type of library; a side-by-side comparison is also presented. PMID:26033846

  13. Selective isolation of G-quadruplexes by affinity chromatography.

    PubMed

    Chang, Tianjun; Liu, Xiangjun; Cheng, Xiaohong; Qi, Cui; Mei, Hongcheng; Shangguan, Dihua

    2012-07-13

    G-quadruplex (G4) is a characteristic secondary structure of nucleic acids containing repetitive tandem guanines. G4-forming sequences are found prevalent in the human genome by bioinformatics analysis. Accumulating evidence has suggested that G4s are involved in many biological processes. Selective isolation of G4s would be an effective tool in the study of G4s. In this paper, we prepared four affinity matrixes using hemin or a perylene derivative (N,N'-Bis-(2-(amino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide, Pery01) as ligand, and investigated the retention behaviors of different G4s on these matrixes. Our experimental results suggest that the π-π stacking interaction between ligand and G-tetrad plays a key role in the selective isolation of G4s, whereas the electrostatic interaction between DNA and matrix causes the nonspecific binding. One matrix prepared by immobilizing Pery01 on polyglycidylmethacrylate (PGMA) beads through an aminocaproic acid spacer exhibits good selectivity for parallel structure G4s and has been successfully used to directly isolate a spiked parallel G4 from plasma. PMID:22398385

  14. Displacement affinity chromatography of protein phosphatase one (PP1) complexes

    PubMed Central

    Moorhead, Greg BG; Trinkle-Mulcahy, Laura; Nimick, Mhairi; De Wever, Veerle; Campbell, David G; Gourlay, Robert; Lam, Yun Wah; Lamond, Angus I

    2008-01-01

    Background Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes. PMID:19000314

  15. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals. PMID:26952369

  16. Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography.

    PubMed

    Müller, Egbert; Vajda, Judith

    2016-05-15

    Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected. PMID:26830536

  17. A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

    PubMed

    Albores, Silvana; Moros, Maria; Cerdeiras, Maria Pia; de la Fuente, Jesus Martinez; Grazu, Valeria; Fraguas, Laura Franco

    2016-01-01

    Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification). PMID:27279446

  18. The derivatization of oxidized polysaccharides for protein immobilization and affinity chromatography.

    PubMed

    Junowicz, E; Charm, S E

    1976-03-25

    The present report describes the preparation of modified polysaccharides matrices useful for the synthesis of affinity adsorbents and immobilized proteins. Hydrazido-matrices were synthesized by condensing an excess of the bifunctional reagent, adipic acid dihydrazide, with periodate oxidized cellulose paper, Sephadex, or Sepharose matrices. Ribonucleotide dialdehyde cofactors, glyceraldehyde 3-phosphate, pyridoxal 5'-phosphate and oxidized DNAase B were separately bound to the hydrazido-polymers. Azido-matrices obtained by modification of the hydrazido-derivatives were coupled to specific amino ligands such as amino acids and proteins. Several adsorbents were prepared and used as models for affinity chromatography. PMID:1260016

  19. Affinity chromatography and affinity labeling of rat liver succinyl-CoA synthetase.

    PubMed

    Ball, D J; Nishimura, J S

    1980-11-25

    Succinyl-CoA synthetase has been purified to apparent homogeneity from rat liver. The key step in the purification procedure involved adsorption on a GDP dialdehyde (dial-GDP)-adipic dihydrazide-Sepharose 4B column and elution by GDP-Mg2+. Like the pig heart enzyme (Brownie, E. R., and Bridger, W. A. (1972) Can. J. Biochem. 50, 719--724), the rat liver enzyme was an alpha beta heterodimer and only the alpha subunit was phosphorylated by [gamma-32P]GTP. The A 280(0.1%) of the enzyme was determined to be 0.5. Amino acid analyses revealed significant similarities in 50% of the amino acid residues of rat liver and Escherichia coli succinyl-CoA synthetases. However, immunodiffusion analysis failed to reveal any antigenic identity between the two enzymes. Incubation with the affinity label, dial-GDP, in the presence of Mg2+ resulted in a biphasic inactivation of the enzyme. The extent of the rapid phase of inactivation appeared to be related to the extent of dephosphorylation of the enzyme and was prevented by preincubation of the enzyme with GTP-Mg2+. The presence of GDP-Mg2+ in the incubation medium prevented the slow phase of the inactivation and retarded the rapid phase. Dephosphorylated enzyme was approximately 2 orders of magnitude more susceptible to inactivation by dial-GDP than phosphorylated enzyme. Labeling of succinyl-CoA synthetase with [3H]dial-GDP gave a linear relationship between inactivation and incorporation of radioactivity with an extrapolated value of less than 1.2 mol of analog/mol of enzyme at 100% inactivation. The distribution of the label in enzyme that was inactivated 40% was approximately 60% in the alpha subunit and 40% in the beta subunit. Thus, while phosphorylation of the enzyme occurs exclusively in the alpha subunit, the nucleotide binding site appears to include components from both alpha and beta subunits. PMID:7430155

  20. Single-step purification of native miraculin using immobilized metal-affinity chromatography.

    PubMed

    Duhita, Narendra; Hiwasa-Tanase, Kyoko; Yoshida, Shigeki; Ezura, Hiroshi

    2009-06-24

    Miraculin is a taste-modifying protein that can be isolated from miracle fruit ( Richadella dulcifica ), a shrub native to West Africa. It is able to turn a sour taste into a sweet taste. The commercial exploitation of this sweetness-modifying protein is underway, and a fast and efficient purification method to extract the protein is needed. We succeeded in purifying miraculin from miracle fruit in a single-step purification using immobilized metal-affinity chromatography (IMAC). The purified miraculin exhibited high purity (>95%) in reverse-phase high-performance liquid chromatography. We also demonstrated the necessity of its structure for binding to the nickel-IMAC column. PMID:19469504

  1. One-step purification of phosphinothricin acetyltransferase using reactive dye-affinity chromatography.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2015-01-01

    Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency. PMID:25749943

  2. Proteomic analysis of human bladder epithelial cells by 2D blue native SDS-PAGE reveals TCDD-induced alterations of calcium and iron homeostasis possibly mediated by nitric oxide.

    PubMed

    Verma, Nisha; Pink, Mario; Petrat, Frank; Rettenmeier, Albert W; Schmitz-Spanke, Simone

    2015-01-01

    A proteomic analysis of the interaction among multiprotein complexes involved in 2,3,7,8-dibenzo-p-dioxin (TCDD)-mediated toxicity in urinary bladder epithelial RT4 cells was performed using two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE). To enrich the protein complexes, unexposed and TCDD-exposed cells were fractionated. BN/SDS-PAGE of the resulting fractions led to an effective separation of proteins and protein complexes of various origins, including cell membrane, mitochondria, and other intracellular compartments. Major differences between the proteome of control and exposed cells involved the alteration of many calcium-regulated proteins (calmodulin, protein S100-A2, annexin A5, annexin A10, gelsolin isoform b) and iron-regulated proteins (ferritin, heme-binding protein 2, transferrin). On the basis of these findings, the intracellular calcium concentration was determined, revealing a significant increase after 24 h of exposure to TCDD. Moreover, the concentration of the labile iron pool (LIP) was also significantly elevated in TCDD-exposed cells. This increase was strongly inhibited by the calmodulin (CaM) antagonist W-7, which pointed toward a possible interaction between iron and calcium signaling. Because nitric oxide (NO) production was significantly enhanced in TCDD-exposed cells and was also inhibited by W-7, we hypothesize that alterations in calcium and iron homeostasis upon exposure to TCDD may be linked through NO generated by CaM-activated nitric oxide synthase. In our model, we propose that NO produced upon TCDD exposure interacts with the iron centers of iron-regulatory proteins (IRPs) that modulate the alteration of ferritin and transferrin, resulting in an augmented cellular LIP and, hence, increased toxicity. PMID:25348606

  3. Antibody purification using affinity chromatography: a case study with a monoclonal antibody to ractopamine.

    PubMed

    Wang, Zhanhui; Liang, Qi; Wen, Kai; Zhang, Suxia; Shen, Jianzhong

    2014-11-15

    The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA). PMID:25261834

  4. Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide

    PubMed Central

    Barry, Standish; O'Carra, Pádraig

    1973-01-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD+ through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD+ (probably through the 8 position of the adenine residue) to a number of different spacer-arm–agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD+ derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD+. Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD+-binding site of this enzyme. Problems

  5. Specific recognition of supercoiled plasmid DNA by affinity chromatography using a synthetic aromatic ligand.

    PubMed

    Caramelo-Nunes, Catarina; Tomaz, Cândida T

    2015-01-01

    Liquid chromatography is the method of choice for the purification of plasmid DNA (pDNA), since it is simple, robust, versatile, and highly reproducible. The most important features of a chromatographic procedure are the use of suitable stationary phases and ligands. As conventional purification protocols are being replaced by more sophisticated and selective procedures, the focus changes toward designing and selecting ligands of high affinity and specificity. In fact, the chemical composition of the chromatographic supports determines the interactions established with the target molecules, allowing their preferential retention over the undesirable ones. Here it is described the selective recognition and purification of supercoiled pDNA by affinity chromatography, using an intercalative molecule (3,8-diamino-6-phenylphenanthridine) as ligand. PMID:25749945

  6. Use of quantitative affinity chromatography for characterizing high-affinity interactions: binding of heparin to antithrombin III.

    PubMed

    Hogg, P J; Jackson, C M; Winzor, D J

    1991-02-01

    The versatility of quantitative affinity chromatography (QAC) for evaluating the binding of macromolecular ligands to macromolecular acceptors has been increased substantially as a result of the derivation of the equations which describe the partitioning of acceptor between matrix-bound and soluble forms in terms of total, rather than free, ligand concentrations. In addition to simplifying the performance of the binding experiments, this development makes possible the application of the technique to systems characterized by affinities higher than those previously amenable to investigation by QAC. Addition of an on-line data acquisition system to monitor the concentration of partitioning solute in the liquid phase as a function of time has permitted the adoption of an empirical approach for determining the liquid-phase concentration of acceptor in the system at partition equilibrium, a development which decreases significantly the time required to obtain a complete binding curve by QAC. The application of these new QAC developments is illustrated by the determination of binding constants for the interactions of high-affinity heparin (Mr 20,300) with antithrombin III at three temperatures. Association constants of 8.0 +/- 2.2 x 10(7), 3.4 +/- 0.3 x 10(7), and 1.0 +/- 0.2 x 10(7) M-1 were observed at 15, 25, and 35 degrees C, respectively. The standard enthalpy change of -4.2 +/- 0.6 kcal/mol that is calculated from these data is in good agreement with a reported value obtained from fluorescence quenching measurements. PMID:2035830

  7. A novel gigaporous GSH affinity medium for high-speed affinity chromatography of GST-tagged proteins.

    PubMed

    Huang, Yongdong; Zhang, Rongyue; Li, Juan; Li, Qiang; Su, Zhiguo; Ma, Guanghui

    2014-03-01

    Novel GSH-AP (phenoxyl agarose coated gigaporous polystyrene, Agap-co-PSt) microspheres were successfully prepared by introducing GSH ligand into hydrophilic AP microspheres pre-activated with 1,4-butanediol diglycidyl ether. The gigaporous structure and chromatographic properties of GSH-AP medium were evaluated and compared with commercial GSH Sepharose FF (GSH-FF) medium. The macropores (100-500nm) of gigaporous PSt microspheres were well maintained after coating with agarose and functionalized with GSH ligand. Hydrodynamic experiments showed that GSH-AP column had less backpressure and plate height than those of GSH-FF column at high flow velocity, which was beneficial for its use in high-speed chromatography. The presence of flow-through pores in GSH-AP microspheres also accelerated the mass transfer rate of biomolecules induced by convective flow, leading to high protein resolution and high dynamic binding capacity (DBC) of glutathione S-transferase (GST) at high flow velocity. High purity of GST and GST-tagged recombinant human interleukin-1 receptor antagonist (rhIL-1RA) were obtained from crude extract with an acceptable recovery yield within 1.5min at a velocity up to 1400cm/h. GSH-AP medium is promising for high-speed affinity chromatography for the purification of GST and GST-tagged proteins. PMID:24269760

  8. Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

    PubMed

    Brgles, Marija; Kurtović, Tihana; Kovačič, Lidija; Križaj, Igor; Barut, Miloš; Lang Balija, Maja; Allmaier, Günter; Marchetti-Deschmann, Martina; Halassy, Beata

    2014-01-01

    In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time. PMID:24217948

  9. Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

    PubMed

    Mönster, Andrea; Hiller, Oliver; Grüger, Daniela; Blasczyk, Rainer; Kasper, Cornelia

    2011-02-01

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column. PMID:21194702

  10. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose.

    PubMed

    Jia, Yinshan; Jarrett, Harry W

    2015-08-01

    The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP-C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3' end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach. PMID:25935261

  11. Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling.

    PubMed

    Kennedy, Jacob J; Yan, Ping; Zhao, Lei; Ivey, Richard G; Voytovich, Uliana J; Moore, Heather D; Lin, Chenwei; Pogosova-Agadjanyan, Era L; Stirewalt, Derek L; Reding, Kerryn W; Whiteaker, Jeffrey R; Paulovich, Amanda G

    2016-02-01

    A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. PMID:26621847

  12. Method for trapping affinity chromatography of transcription factors using aldehyde-hydrazide coupling to agarose

    PubMed Central

    Jia, Yinshan; Jarrett, Harry W.

    2015-01-01

    The uses of a method of coupling DNA is investigated for trapping and purifying transcription factors. Using the GFP-C/EBP fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry utilized is mild and was shown to have no detrimental effect on GFP fluorescence or GFP-C/EBP DNA-binding. The method involves introducing a ribose nucleotide to the 3′ end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose which couples to hydrazide-agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture and DNA-protein complexes form. The complex is then coupled to hydrazide-agarose for trapping the DNA-protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes including E2A, c-myc, and myo-D were also purified but myogenenin and NFκB were not. Therfore, this approach proved valuable for both affinity chromatography and for the trapping approach. PMID:25935261

  13. Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

    PubMed Central

    Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

    2013-01-01

    The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

  14. Evaluation of capillary chromatographic supports for immobilized human purine nucleoside phosphorylase in frontal affinity chromatography studies.

    PubMed

    de Moraes, Marcela Cristina; Temporini, Caterina; Calleri, Enrica; Bruni, Giovanna; Ducati, Rodrigo Gay; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra; Massolini, Gabriella

    2014-04-18

    The aim of this work was to optimize the preparation of a capillary human purine nucleoside phosphorylase (HsPNP) immobilized enzyme reactor (IMER) for characterization and affinity screening studies of new inhibitors by frontal affinity chromatography coupled to mass spectrometry (FAC-MS). For this purpose two monolithic supports, a Chromolith Speed Rod (0.1mm I.D.×5cm) and a methacrylate-based monolithic epoxy polymeric capillary column (0.25mm I.D.×5cm) with epoxy reactive groups were considered and compared to an IMER previously developed using an open fused silica capillary. Each HsPNP-IMER was characterized in terms of catalytic activity using Inosine as standard substrate. Furthermore, they were also explored for affinity ranking experiments. Kd determination was carried out with the based fused silica HsPNP-IMER and the results are herein discussed. PMID:24630982

  15. Synthesis and application of a new cleavable linker for "click"-based affinity chromatography.

    PubMed

    Landi, Felicetta; Johansson, Conny M; Campopiano, Dominic J; Hulme, Alison N

    2010-01-01

    A new chemically-cleavable linker has been synthesised for the affinity-independent elution of biomolecules by classical affinity chromatography. This azo-based linker is shown to couple efficiently with "click" derivatised ligands such as biotin propargyl amide through a copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition reaction. Binding to Affi-Gel matrices displaying ligands coupled to the new linker is both efficient and selective. The captured material may be readily released from the resin upon treatment with sodium dithionite. These mild elution conditions have allowed for the efficient isolation of the affinity partner from complex protein mixtures such as those found in fetal bovine serum. PMID:20024132

  16. A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

    PubMed Central

    Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

    2013-01-01

    Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

  17. Purification of proteins containing zinc finger domains using Immobilized Metal Ion Affinity Chromatography

    PubMed Central

    Voráčková, Irena; Suchanová, Šárka; Ulbrich, Pavel; Diehl, William E.; Ruml, Tomáš

    2011-01-01

    Heterologous proteins are frequently purified by Immobilized Metal Ion Affinity Chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e. CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state. PMID:21600288

  18. Protecting group-free immobilization of glycans for affinity chromatography using glycosylsulfonohydrazide donors.

    PubMed

    Hernandez Armada, Daniel; Santos, Jobette T; Richards, Michele R; Cairo, Christopher W

    2015-11-19

    A variety of applications in glycobiology exploit affinity chromatography through the immobilization of glycans to a solid support. Although several strategies are known, they may provide certain advantages or disadvantages in how the sugar is attached to the affinity matrix. Additionally, the products of some methods may be hard to characterize chemically due to non-specific reactions. The lack of specificity in standard immobilization reactions makes affinity chromatography with expensive oligosaccharides challenging. As a result, methods for specific and efficient immobilization of oligosaccharides remain of interest. Herein, we present a method for the immobilization of saccharides using N'-glycosylsulfonohydrazide (GSH) carbohydrate donors. We have compared GSH immobilization to known strategies, including the use of divinyl sulfone (DVS) and cyanuric chloride (CC), for the generation of affinity matrices. We compared immobilization methods by determining their immobilization efficiency, based on a comparison of the mass of immobilized carbohydrate and the concentration of active binding sites (determined using lectins). Our results indicate that immobilization using GSH donors can provide comparable amounts of carbohydrate epitopes on solid support while consuming almost half of the material required for DVS immobilization. The lectin binding capacity observed for these two methods suggests that GSH immobilization is more efficient. We propose that this method of oligosaccharide immobilization will be an important tool for glycobiologists working with precious glycan samples purified from biological sources. PMID:26454791

  19. Purification to homogeneity of an active opioid receptor from rat brain by affinity chromatography.

    PubMed

    Loukas, S; Mercouris, M; Panetsos, F; Zioudrou, C

    1994-05-10

    Active opioid binding proteins were solubilized from rat brain membranes in high yield with sodium deoxycholate in the presence of NaCl. Purification of opioid binding proteins was accomplished by opioid antagonist affinity chromatography. Chromatography using the delta-opioid antagonist N,N-diallyl-Tyr-D-Leu-Gly-Tyr-Leu attached to omega-aminododecyl-agarose (Affi-G) (procedure A) yielded a partially purified protein that binds selectively the delta-opioid agonist [3H]Tyr-D-Ser-Gly-Phe-Leu-Thr ([3H]DSLET), with a Kd of 19 +/- 3 nM and a Bmax of 5.1 +/- 0.4 nmol/mg of protein. Subsequently, Lens culinaris agglutinin-Sepharose 4B chromatography of the Affi-G eluate resulted in isolation of an electrophoretically homogeneous protein of 58 kDa that binds selectively [3H]DSLET with a Kd of 21 +/- 3 nM and a Bmax of 16.5 +/- 1.0 nmol/mg of protein. Chromatography using the nonselective antagonist 6-aminonaloxone coupled to 6-aminohexanoic acid-Sepharose 4B (Affi-NAL) (procedure B) resulted in isolation of a protein that binds selectively [3H]DSLET with a Kd of 32 +/- 2 nM and a Bmax of 12.4 +/- 0.5 nmol/mg of protein, and NaDodSO4/PAGE revealed a major band of apparent molecular mass 58 kDa. Polyclonal antibodies (Anti-R IgG) raised against the Affi-NAL protein inhibit the specific [3H]DSLET binding to the Affi-NAL eluate and to the solubilized membranes. Moreover, the Anti-R IgG inhibits the specific binding of radiolabeled Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO; mu-agonist), DSLET (delta-agonist), and naloxone to homogenates of rat brain membranes with equal potency. Furthermore, immunoaffinity chromatography of solubilized membranes resulted in the retention of a major protein of apparent molecular mass 58 kDa. In addition, immunoblotting of solubilized membranes and purified proteins from the Affi-G and Affi-NAL matrices revealed that the Anti-R IgG interacts with a protein of 58 kDa. PMID:8183950

  20. Partial purification of the 5-hydroxytryptophan-reuptake system from human blood platelets using a citalopram-derived affinity resin

    SciTech Connect

    Biessen, E.A.L; Horn, A.S.; Robillard, G.T. )

    1990-04-03

    This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50-70-fold increase in specific ({sup 3}H) imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 {mu}M citalopram resulted in a 22% recovery of binding activity. A 10,000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after {sup 125}I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of ({sup 3}H) imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and ({sup 3}H)imipramine binding activity.

  1. Characteristics of the interaction of calcium with casein submicelles as determined by analytical affinity chromatography

    SciTech Connect

    Jang, H.D.; Swaisgood, H.E. )

    1990-12-01

    Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions.

  2. Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

    SciTech Connect

    Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.; Chen, J.S.; Wogan, G.N.

    1985-10-01

    A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with ( UC)aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.

  3. Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme.

    PubMed

    Cass, Brian; Pham, Phuong Lan; Kamen, Amine; Durocher, Yves

    2005-03-01

    Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%. PMID:15721774

  4. Development and Validation of an Affinity Chromatography-Protein G Method for IgG Quantification

    PubMed Central

    Paradina Fernández, Lesly; Calvo, Loany; Viña, Lisel

    2014-01-01

    Nimotuzumab, an IgG that recognizes the epidermal growth factor receptor (EGF-R) overexpressed in some tumors, is used in the treatment of advanced head and neck cancer. For the quantification of this protein in cell culture supernatants, protein G-HPLC affinity chromatography is used due to its high affinity and specificity for antibodies of this class. The technique relies on the comparison of the area under the curve of the elution peak of the samples to be evaluated versus to a calibration curve of well-known concentrations and was validated by assessment of its robustness, specificity, repeatability, intermediate precision, accuracy, linearity, limit of detection, limit of quantification, and range. According to results of the study all validation parameters fulfilled the preestablished acceptance criteria and demonstrated the feasibility of the assay for the analysis of samples of cell culture supernatant as well as drug product. PMID:27379284

  5. Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins.

    PubMed

    Duong-Thi, Minh-Dao; Bergström, Maria; Edwards, Katarina; Eriksson, Jonny; Ohlson, Sten; Ying, Janet To Yiu; Torres, Jaume; Hernández, Víctor Agmo

    2016-02-01

    Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures. PMID:26673836

  6. Procedure for rapid isolation of photosynthetic reaction centers using cytochrome c affinity chromatography

    SciTech Connect

    Brudvig, G.W.; Worland, S.T.; Sauer, K.

    1983-02-01

    Horse heart cytochrome c linked to Sepharose 4B is used to purify reaction centers from Rhodopseudomonas sphaeroides R-26. This procedure allows for an initial recovery of 80-90% of the bacterial reaction centers present in chromatophore membranes. High purity reaction centers (A/sub 280//A/sub 802/ < 1.30) can be obtained with a 30% recovery. Reaction centers from wild-type Rps. sphaeroides and Rps. capsulata also bind to a cytochrome c column. Cytochrome c affinity chromatography can also be used to isolate photosystem I complexes from spinach chloroplasts.

  7. Fractionation of Aspergillus niger cellulases by combined ion exchange affinity chromatography

    SciTech Connect

    Boyer, R.F.; Allen, T.L.; Dykema, P.A.

    1987-02-05

    Eight chemically modified cellulose supports were tested for their ability to adsorb components of the Aspergillus niger cellulase system. At least two of the most effective adsorbents, aminoethyl cellulose and carboxymethyl cellulose, were shown to be useful for the fractionation of cellulases. These supports apparently owe their resolving capacity to both ion exchange and biospecific binding effects; however, the relative importance of each effect is unknown. These observations form the basis for a new cellulase fractionation technique, combined ion exchange-affinity chromatography. 22 references.

  8. Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli.

    PubMed

    Velkov, Tony; Jones, Alun; Lim, Maria L R

    2008-01-01

    A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni(2+)-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor. PMID:18800304

  9. Development of a novel affinity chromatography resin for platform purification of lambda fabs.

    PubMed

    Eifler, Nora; Medaglia, Giovanni; Anderka, Oliver; Laurin, Linus; Hermans, Pim

    2014-01-01

    Antigen-binding fragments (Fabs) are novel formats in the growing pipeline of biotherapeutics. Sharing similar features to monoclonal antibodies (mAbs) with regard to expression, Fabs are considered as unchallenging for upstream development. Yet for downstream processing, the mature mAb downstream purification platform is not directly applicable. New approaches need to be found to achieve a lean purification process that maintains quality, productivity, and timelines while being generically applicable independent of the expression system. In a successful collaboration, BAC BV, GE Healthcare, and Novartis Pharma AG have developed a new affinity chromatography medium (resin) suitable to support cGMP manufacturing of lambda Fabs. We show that using this novel chromatography medium for the capture step, a purification platform for lambda Fabs can be established. PMID:25082738

  10. Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography.

    PubMed

    Yatsukawa, Yoh-Ichi; Ito, Hironobu; Matsuda, Takahiro; Nakamura, Munetomo; Watai, Masatoshi; Fujita, Kazuhiro

    2011-01-01

    A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively. PMID:21919363

  11. Development of an aptamer-affinity chromatography for efficient single step purification of Concanavalin A from Canavalia ensiformis.

    PubMed

    Ahirwar, Rajesh; Nahar, Pradip

    2015-08-01

    Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41ntssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13mm, 20mm and 25mm diameter columns having a bed height of 60mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A. PMID:26102634

  12. Analysis of membrane-protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native-PAGE complexome profiling and 2D blue native-/SDS-PAGE.

    PubMed

    Wöhlbrand, Lars; Ruppersberg, Hanna S; Feenders, Christoph; Blasius, Bernd; Braun, Hans-Peter; Rabus, Ralf

    2016-03-01

    Sulfate-reducing bacteria (SRB) obtain energy from cytoplasmic reduction of sulfate to sulfide involving APS-reductase (AprAB) and dissimilatory sulfite reductase (DsrAB). These enzymes are predicted to obtain electrons from membrane redox complexes, i.e. the quinone-interacting membrane-bound oxidoreductase (QmoABC) and DsrMKJOP complexes. In addition to these conserved complexes, the genomes of SRB encode a large number of other (predicted) membrane redox complexes, the function and actual formation of which is unknown. This study reports the establishment of 1D Blue Native-PAGE complexome profiling and 2D BN-/SDS-PAGE for analysis of the membrane protein complexome of the marine sulfate reducer Desulfobacula toluolica Tol2. Analysis of normalized score profiles of >800 proteins in combination with hierarchical clustering and identification of 2D BN-/SDS-PAGE separated spots demonstrated separation of membrane complexes in their native form, e.g. ATP synthase. In addition to the QmoABC and DsrMKJOP complexes, other complexes were detected that constitute the basic membrane complexome of D. toluolica Tol2, e.g. transport proteins (e.g. sodium/sulfate symporters) or redox complexes involved in Na(+) -based bioenergetics (RnfABCDEG). Notably, size estimation indicates dimer and quadruple formation of the DsrMKJOP complex in vivo. Furthermore, cluster analysis suggests interaction of this complex with a rhodanese-like protein (Tol2_C05230) possibly representing a periplasmic electron transfer partner for DsrMKJOP. PMID:26792001

  13. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  14. Affinity chromatography reveals RuBisCO as an ecdysteroid-binding protein.

    PubMed

    Uhlik, Ondrej; Kamlar, Marek; Kohout, Ladislav; Jezek, Rudolf; Harmatha, Juraj; Macek, Tomas

    2008-12-22

    The aim of this work was to isolate plant ecdysteroid-binding proteins using affinity chromatography. Ecdysteroids as insect hormones have been investigated thoroughly but their function and the mechanism of action in plants and other organisms is still unknown although ecdysteroids occur in some plants in a relatively large amount. Therefore, 20-hydroxyecdysone was immobilized on a polymeric carrier as a ligand for affinity chromatography in order to isolate plant ecdysteroid-binding proteins from the cytosolic extract of New Zealand spinach (Tetragonia tetragonoides). Non-specifically bound proteins were eluted with a rising gradient of concentration of sodium chloride, and 3% (v/v) acetic acid was used for the elution of the specifically bound proteins. Using this method, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was isolated. The influence of ecdysteroids on RuBisCO was further studied. Our results show that ecdysteroids are able to increase the yield of RuBisCO-mediated reaction in which CO(2) is fixed into organic matter by more than 10%. PMID:18761365

  15. Purification of 3-phosphoglycerate kinase from diverse sources by affinity elution chromatography.

    PubMed Central

    Fifis, T; Scopes, R K

    1978-01-01

    1. Affinity elution chromatography was used to purify phosphoglycerate kinase from a variety of sources. The choice of buffer pH for the chromatography was made according to the relative electrophoretic mobility of the enzyme from the species concerned. 2. Outlines of the methods used to isolate the enzyme from over 20 sources are presented. The enzyme was purified from the muscle tissue of a variety of mammals, fish and birds, from liver of several animals, from yeast, Escherichia coli, and plant leaves. The more acidic varieties of the enzymes were purified by conventional gradient elution from ion-exchangers as affinity elution procedures were not applicable. 3. The structural and kinetic parameters investigated show that phosphoglycerate kinase is evolutionarily a highly conservative enzyme; there were few differences in properties regardless of source or function (glycolytic, gluconeogenic or photosynthetic). 4. A detailed comparison of the enzyme preparations purified from bovine muscle and bovine liver failed to detect any significant differences between them; the evidence indicates that they are genetically identical. PMID:367367

  16. Characterization of Murine Brain Membrane Glycoproteins by Detergent Assisted Lectin Affinity Chromatography (DALAC)

    PubMed Central

    Wei, Xin; Dulberger, Charles; Li, Lingjun

    2010-01-01

    Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization has been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that under certain concentrations, detergents can minimize the nonspecific bindings and facilitate the elution of hydrophobic glycoproteins. With the Detergent Assisted Lectin Affinity Chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. 699 proteins were identified with at least two unique peptides, of which 219 are membrane glycoproteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome. PMID:20700909

  17. Determination of the kinetic rate constant of cyclodextrin supramolecular systems by high-performance affinity chromatography.

    PubMed

    Zhang, Jiwen; Li, Haiyan; Sun, Lixin; Wang, Caifen

    2015-01-01

    The kinetics of the association and dissociation are fundamental kinetic processes for the host-guest interactions (such as the drug-target and drug-excipient interactions) and the in vivo performance of supramolecules. With advantages of rapid speed, high precision and ease of automation, the high-performance affinity chromatography (HPAC) is one of the best techniques to measure the interaction kinetics of weak to moderate affinities, such as the typical host-guest interactions of drug and cyclodextrins by using a cyclodextrin-immobilized column. The measurement involves the equilibration of the cyclodextrin column, the upload and elution of the samples (non-retained substances and retained solutes) at different flow rates on the cyclodextrin and control column, and data analysis. It has been indicated that cyclodextrin-immobilized chromatography is a cost-efficient high-throughput tool for the measurement of (small molecule) drug-cyclodextrin interactions as well as the dissociation of other supramolecules with relatively weak, fast, and extensive interactions. PMID:25749964

  18. p53-Encoding pDNA Purification by Affinity Chromatography for Cancer Therapy.

    PubMed

    Sousa, Ângela; Queiroz, João A; Sousa, Fani

    2015-01-01

    The gene therapy approach based on reestablishment of p53 tumor suppressor, which acts as a prevailing guardian against malignant cell transformation, is raising new prospects on the outcome of an effective anticancer treatment. It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufacturing process. Therefore, several downstream methods have been proposed to achieve high quantities of supercoiled plasmid DNA with pharmaceutical grade purity. Affinity chromatography with amino acids as ligands has recently yielded interesting results because these ligands take advantage of their biological function or chemical structure to promote specific interactions with different nucleic acids. Here, we describe detailed procedures for the preparation and purification of supercoiled plasmid DNA, with the purity degree required by regulatory agencies, by using arginine affinity chromatography. With this methodology pure pDNA is obtained, efficient on eukaryotic cell transfection and biologically active, resulting in the reestablishment of the p53 protein levels in cancer cell lines. PMID:26072404

  19. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    PubMed Central

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  20. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies.

    PubMed

    Boulet-Audet, Maxime; Kazarian, Sergei G; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  1. Affinity purification of heme-tagged proteins.

    PubMed

    Asher, Wesley B; Bren, Kara L

    2014-01-01

    Protein affinity purification techniques are widely used for isolating pure target proteins for biochemical and structural characterization. Herein, we describe the protocol for affinity-based purification of proteins expressed in Escherichia coli that uses the coordination of a peptide tag covalently modified with heme c, known as a heme-tag, to an L-histidine immobilized Sepharose resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. In addition, we describe methods for specifically detecting heme-tagged proteins in SDS-PAGE gels using a heme-staining procedure and for quantifying the proteins using a pyridine hemochrome assay. PMID:24943311

  2. Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

    PubMed

    Meiby, Elinor; Knapp, Stefan; Elkins, Jonathan M; Ohlson, Sten

    2012-11-01

    Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential. PMID:22918538

  3. High-performance affinity monolith chromatography for chiral separation and determination of enzyme kinetic constants.

    PubMed

    Yao, Chunhe; Qi, Li; Qiao, Juan; Zhang, Haizhi; Wang, Fuyi; Chen, Yi; Yang, Gengliang

    2010-09-15

    A new kind of immobilized human serum albumin (HSA) column was developed by using the sub-micron skeletal polymer monolith based on poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-EDMA)] as the support of high-performance affinity chromatography. Using the epoxide functional groups presented in GMA, the HSA immobilization procedure was performed by two different means. The affinity columns were successfully adopted for the chiral separation of D,L-amino acids (AAs). Then this method was shown to be applicable to the quantitative analysis of D-tryptophan, with a linear range between 12.0 microM and 979.0 microM, and a correlation coefficient above 0.99. Furthermore, it was used for the analysis of urine sample. This assay is demonstrated to be facile and relatively rapid. So it allows us to measure the enzyme catalytic activity in the incubation of D,L-AAs with D-AA oxidase and to study the kinetics of the enzyme reaction. It implied that the affinity monolithic columns can be a useful tool for studying DAAO enzyme reaction and investigating the potential enzyme mechanism requirement among chiral conversion. PMID:20801337

  4. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.

    PubMed

    Habicht, K-L; Singh, N S; Indig, F E; Wainer, I W; Moaddel, R; Shimmo, R

    2015-09-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (Kd) determined were 1.08±0.49 and 0.0086±0.0006μM, respectively, consistent with previously reported values. Furthermore, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  5. Affinity chromatography of chaperones based on denatured proteins: Analysis of cell lysates of different origin.

    PubMed

    Marchenko, N Yu; Sikorskaya, E V; Marchenkov, V V; Kashparov, I A; Semisotnov, G V

    2016-03-01

    Molecular chaperones are involved in folding, oligomerization, transport, and degradation of numerous cellular proteins. Most of chaperones are heat-shock proteins (HSPs). A number of diseases of various organisms are accompanied by changes in the structure and functional activity of chaperones, thereby revealing their vital importance. One of the fundamental properties of chaperones is their ability to bind polypeptides lacking a rigid spatial structure. Here, we demonstrate that affinity chromatography using sorbents with covalently attached denatured proteins allows effective purification and quantitative assessment of their bound protein partners. Using pure Escherichia coli chaperone GroEL (Hsp60), the capacity of denatured pepsin or lysozyme-based affinity sorbents was evaluated as 1 mg and 1.4 mg of GroEL per 1 ml of sorbent, respectively. Cell lysates of bacteria (E. coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as the lysate of rat liver mitochondria were analyzed using affinity carrier with denatured lysozyme. It was found that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme. PMID:26644295

  6. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  7. ANALYSIS OF DRUG INTERACTIONS WITH HIGH DENSITY LIPOPROTEIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Chen, Sike; Sobansky, Matthew R.; Hage, David S.

    2009-01-01

    Columns containing immobilized lipoproteins were prepared for the analysis of drug interactions with these particles by high-performance affinity chromatography. This approach was evaluated by using it to examine the binding of high density lipoprotein (HDL) to the drugs propranolol or verapamil. HDL was immobilized by the Schiff base method onto silica and gave HPLC columns with reproducible binding to propranolol over four to five days of continuous operation at pH 7.4. Frontal analysis experiments indicated that two types of interactions were occurring between R/S-propranolol and HDL at 37°C: saturable binding with an association equilibrium constant (Ka) of 1.1–1.9 × 105 M−1, and non-saturable binding with an overall affinity constant (n Ka) of 3.7–4.1 × 104 M−1. Similar results were found at 4 and 27°C. Verapamil also gave similar behavior, with a Ka of 6.0 × 104 M−1 at 37°C for the saturable sites and a n Ka value for the non-saturable sites of 2.5 × 104 M−1. These measured affinities gave good agreement with solution-phase values. The results indicated HPAC can be used to study drug interactions with HDL, providing information that should be valuable in obtaining a better description of how drugs are transported within the body. PMID:19833090

  8. Weak affinity chromatography as a new approach for fragment screening in drug discovery.

    PubMed

    Duong-Thi, Minh-Dao; Meiby, Elinor; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2011-07-01

    Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM-μM in dissociation constant (K(D))) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23-compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM-10 μM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC-MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening. PMID:21352794

  9. SwellGel: a sample preparation affinity chromatography technology for high throughput proteomic applications.

    PubMed

    Haney, Paul J; Draveling, Connie; Durski, Wendy; Romanowich, Kathryn; Qoronfleh, M Walid

    2003-04-01

    Development of high throughput systems for purification and analysis of proteins is essential for the success of today's proteomic research. We have developed an affinity chromatography technology that allows the customization of high capacity/high throughput chromatographic separation of proteins. This technology utilizes selected chromatography media that are dehydrated to form uniform SwellGel discs. Unlike wet resin slurries, these discs are easily adaptable to a variety of custom formats, eliminating problems associated with resin dispensing, equilibration, or leakage. Discs can be made in assorted sizes (resin volume 15 microl-3 ml) dispensed in various formats (384-, 96-, 48-, and 24-well microplates or columns) and different ligands can be attached to the matrix. SwellGel discs rapidly hydrate upon addition of either water or the protein sample, providing dramatically increased capacity compared to coated plates. At the same time, the discs offer greater stability, reproducibility, and ease of handling than standard wet chromatography resins. We previously reported the development of SwellGel for the purification of 6x His- and glutathione-S-transferase (GST)-tagged fusion proteins [Prot. Exp. Purif. 22 (2001) 359-366]. In this paper, we discuss an expanded list of SwellGel stabilized chromatographic methods that have been adapted to high throughput formats for processing protein samples ranging from 10 microl to 10 ml (1 microg to 50 mg protein). Data are presented applying SwellGel discs to high throughput proteomic applications such as affinity tag purification, protein desalting, the removal of abundant proteins from serum including albumin and immunoglobulin, and the isolation of phosphorylated peptides for mass spectrometry. PMID:12699691

  10. A fullerene C60-based ligand in a stationary phase for affine chromatography of membrane porphyrin-binding proteins

    NASA Astrophysics Data System (ADS)

    Amirshakhi, N.; Alyautdin, R. N.; Orlov, A. P.; Poloznikov, A. A.; Kuznetsov, D. A.

    2008-11-01

    A new affine chromatography technique is suggested for the purification of porphyrin-binding proteins (PBP) from mammal cell membranes. The procedure uses new fullerene-porphyrin ligands immobilized on agarose and bound to the polysaccharide matrix via the epoxycyclohexyl residue. A selective PBP stationary phase was used in a single-column chromatography run for the complete purification of a monomeric protein (17.6 kDa) from mitochondrial membranes of rat myocardium. This protein was characterized by high affinity for porphyrin-related structures. To separate it from other nonspecifically sorbed membrane proteins, synchronous linear pH and ionic strength gradients were used.

  11. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  12. A new type of metal chelate affinity chromatography using trivalent lanthanide ions for phosphopeptide enrichment.

    PubMed

    Mirza, Munazza R; Rainer, Matthias; Messner, Christoph B; Güzel, Yüksel; Schemeth, Dieter; Stasyk, Taras; Choudhary, Muhammad I; Huber, Lukas A; Rode, Bernd M; Bonn, Günther K

    2013-05-21

    In this study, a new type of immobilized metal-ion affinity chromatography (IMAC) resin for the isolation of phosphopeptides was synthesized which is based on the specific interaction between phosphate groups and chelated lanthanide metal ions. In this regard trivalent lanthanum, holmium and erbium ions were chelated to a highly porous phosphonate polymer which was prepared by radical polymerization of vinylphosphonic acid (VPA) and divinylbenzene (DVB). The developed method was evaluated with peptide mixtures from digested standard proteins (α-casein, β-casein and ovalbumin) as well as with bovine milk, egg white and a spiked HeLa cell lysate. Compared to the commonly used TiO2 approach, the presented method showed higher selectivity for phosphorylated peptides. This can be explained by the strong preference of trivalent lanthanide ions for phosphates with which they form very tight ionic bonds. Mono- and multiply phosphorylated peptides could be enriched and released in a single basic elution step, while non-phosphorylated peptides remained on the resin. Ab initio quantum mechanical energy minimizations of model complexes for polymer-ion-ligand interactions provided geometries, binding energies and charges which are discussed in conjunction with the observed experimental properties, leading to the most satisfying agreement. The presented lanthanide-IMAC resins represent promising affinity materials for the selective isolation of phosphopeptides from biological samples. PMID:23552617

  13. Removal of PCR Error Products and Unincorporated Primers by Metal-Chelate Affinity Chromatography

    PubMed Central

    Kanakaraj, Indhu; Jewell, David L.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2011-01-01

    Immobilized Metal Affinity Chromatography (IMAC) has been used for decades to purify proteins on the basis of amino acid content, especially surface-exposed histidines and “histidine tags” genetically added to recombinant proteins. We and others have extended the use of IMAC to purification of nucleic acids via interactions with the nucleotide bases, especially purines, of single-stranded RNA and DNA. We also have demonstrated the purification of plasmid DNA from contaminating genomic DNA by IMAC capture of selectively-denatured genomic DNA. Here we describe an efficient method of purifying PCR products by specifically removing error products, excess primers, and unincorporated dNTPs from PCR product mixtures using flow-through metal-chelate affinity adsorption. By flowing a PCR product mixture through a Cu2+-iminodiacetic acid (IDA) agarose spin column, 94–99% of the dNTPs and nearly all the primers can be removed. Many of the error products commonly formed by Taq polymerase also are removed. Sequencing of the IMAC-processed PCR product gave base-calling accuracy comparable to that obtained with a commercial PCR product purification method. The results show that IMAC matrices (specifically Cu2+-IDA agarose) can be used for the purification of PCR products. Due to the generality of the base-specific mechanism of adsorption, IMAC matrices may also be used in the purification of oligonucleotides, cDNA, mRNA and micro RNAs. PMID:21264292

  14. High-Performance Affinity Chromatography: Applications in Drug-Protein Binding Studies and Personalized Medicine.

    PubMed

    Li, Zhao; Beeram, Sandya R; Bi, Cong; Suresh, D; Zheng, Xiwei; Hage, David S

    2016-01-01

    The binding of drugs with proteins and other agents in serum is of interest in personalized medicine because this process can affect the dosage and action of drugs. The extent of this binding may also vary with a given disease state. These interactions may involve serum proteins, such as human serum albumin or α1-acid glycoprotein, or other agents, such as lipoproteins. High-performance affinity chromatography (HPAC) is a tool that has received increasing interest as a means for studying these interactions. This review discusses the general principles of HPAC and the various approaches that have been used in this technique to examine drug-protein binding and in work related to personalized medicine. These approaches include frontal analysis and zonal elution, as well as peak decay analysis, ultrafast affinity extraction, and chromatographic immunoassays. The operation of each method is described and examples of applications for these techniques are provided. The type of information that can be obtained by these methods is also discussed, as related to the analysis of drug-protein binding and the study of clinical or pharmaceutical samples. PMID:26827600

  15. ANALYSIS OF DRUG INTERACTIONS WITH VERY LOW DENSITY LIPOPROTEIN BY HIGH PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Sobansky, Matthew R.; Hage, David S.

    2014-01-01

    High-performance affinity chromatography (HPAC) was utilized to examine the binding of very low density lipoprotein (VLDL) with drugs, using R/S-propranolol as a model. These studies indicated that two mechanisms existed for the binding of R- and S-propranolol with VLDL. The first mechanism involved non-saturable partitioning of these drugs with VLDL, which probably occurred with the lipoprotein's non-polar core. This partitioning was described by overall affinity constants of 1.2 (± 0.3) × 106 M-1 for R-propranolol and 2.4 (± 0.6) × 106 M-1 for S-propranolol at pH 7.4 and 37 °C. The second mechanism occurred through saturable binding by these drugs at fixed sites on VLDL, such as represented by apolipoproteins on the surface of the lipoprotein. The association equilibrium constants for this saturable binding at 37 °C were 7.0 (± 2.3) × 104 M-1 for R-propranolol and 9.6 (± 2.2) × 104 M-1 for S-propranolol. Comparable results were obtained at 20 °C and 27 °C for the propranolol enantiomers. This work provided fundamental information on the processes involved in the binding of R- and S-propranolol to VLDL, while also illustrating how HPAC can be used to evaluate relatively complex interactions between agents such as VLDL and drugs or other solutes. PMID:25103529

  16. Binding of angiogenesis inhibitor kringle 5 to its specific ligands by frontal affinity chromatography.

    PubMed

    Bian, Liujiao; Li, Qian; Ji, Xu

    2015-07-01

    The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and L-lysine to Kringle 5 were 19.0×10(3), 7.97×10(3), 6.45×10(3), 6.07×10(3) and 4.04×10(3) L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands. PMID:25981289

  17. DETECTION OF HETEROGENEOUS DRUG-PROTEIN BINDING BY FRONTAL ANALYSIS AND HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Tong, Zenghan; Joseph, K.S.; Hage, David S.

    2011-01-01

    This study examined the use of frontal analysis and high-performance affinity chromatography for detecting heterogeneous binding in biomolecular interactions, using the binding of acetohexamide with human serum albumin (HSA) as a model. It was found through the use of this model system and chromatographic theory that double-reciprocal plots could be used more easily than traditional isotherms for the initial detection of binding site heterogeneity. The deviations from linearity that were seen in double-reciprocal plots as a result of heterogeneity were a function of the analyte concentration, the relative affinities of the binding sites in the system and the amount of each type of site that was present. The size of these deviations was determined and compared under various conditions. Plots were also generated to show what experimental conditions would be needed to observe these deviations for general heterogeneous systems or for cases in which some preliminary information was available on the extent of binding heterogeneity. The methods developed in this work for the detection of binding heterogeneity are not limited to drug interactions with HSA but could be applied to other types of drug-protein binding or to additional biological systems with heterogeneous binding. PMID:21612784

  18. Glycan-specific whole cell affinity chromatography: A versatile microbial adhesion platform

    PubMed Central

    Van Tassell, Maxwell L.; Price, Neil P.J.; Miller, Michael J.

    2014-01-01

    We have sought a universal platform for elucidating and exploiting specificity of glycan-mediated adhesion by potentially uncharacterized microorganisms. Several techniques exist to explore microbial interactions with carbohydrate structures. Many are unsuitable for investigating specific mechanisms or uncharacterized organisms, requiring pure cultures, labeling techniques, expensive equipment, or other limitations such as questionable stability, stereospecificity, or scalability. We have adapted an affinity chromatography resin as a model to overcome these drawbacks, among others. It readily allows for the quantification, selection, and manipulation of target organisms based on interactions with glycan ligands. To maximize its utility as a selective screening method, we have constructed the tool such that it:•Promotes whole-cell interactions using viable, unaltered cells.•Provides robust spatial interactions with target glycans, presented with controlled stereo-specificity, for high affinity/avidity interactions that reflect a complex in vivo matrix.•Has the ability to utilize any reducing glycan, is quick, efficient, safe, and affordable to construct, and is scalable and reusable for multiple applications. PMID:26150959

  19. Necator americanus secretory acetylcholinesterase and its purification from excretory-secretory products by affinity chromatography.

    PubMed

    Pritchard, D I; Leggett, K V; Rogan, M T; McKean, P G; Brown, A

    1991-03-01

    Acetylcholinesterase (AChE) secretion by adult N. americanus was enhanced in vitro by incorporating insoluble collagen rafts into culture dishes. Enzyme produced in this way had preferential substrate specificity for acetylthiocholine iodide (ATC), and its activity was inhibited by eserine (1.1 x 10(-8) M). Ancylostoma ceylanicum, another hookworm species, failed to produce comparable amounts of AChE in culture. AChE was efficiently purified from culture medium by affinity chromatography on edrophonium sepharose; 81% of the AChE activity was retained by the affinity matrix, although this fraction contained only 4.3% of the protein loaded. Antisera raised against purified AChE in rabbits immunohistochemically stained the oesophageal glands of the parasite, and reacted with molecules of 32, 60, 80, 140 and 220 kDa in reduced adult ES products on Western blotting, although differential activity was observed against worm homogenates and earlier developmental stages. On IEF, purified AChE resolved predominantly with a pl of 3.55; proteins with a similar pl were recognized by rabbit anti-AChE. IgG preparations of this antiserum inhibited AChE activity in ES products, and inhibited AChE secretion by adult worms in culture. The availability of this immunological probe will allow definitive experiments to be conducted on the role of this enigmatic enzyme in the host-parasite relationship. PMID:2052405

  20. CHARACTERIZATION OF THE BINDING OF SULFONYLUREA DRUGS TO HSA BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Joseph, K.S.; Hage, David S.

    2010-01-01

    Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (± 0.2) × 105 M−1 and 3.5 (± 3.0) × 102 M−1 for acetohexamide and values of 8.7 (± 0.6) × 104 and 8.1 (± 1.7) × 103 M−1 for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (± 0.1) × 105 and 4.3 (± 0.3) × 104 M−1, respectively, at 37°C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (± 0.2) × 104 and 5.3 (± 0.2) × 104 M−1, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions. PMID:20435530

  1. Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

    PubMed

    Salehi, Nasrin; Peng, Ching-An

    2016-07-01

    CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. PMID:27110670

  2. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  3. Separation of TFIIIC into two functional components by sequence specific DNA affinity chromatography.

    PubMed Central

    Dean, N; Berk, A J

    1987-01-01

    Recently, it has been shown that mammalian transcription factor IIIC (TFIIIC) activity can be separated by anion exchange FPLC chromatography into two functional components (1), both of which are required for transcription of tRNA and the adenovirus VA RNA genes. Here we show that these two functional components, designated TFIIIC1 and TFIIIC2, can also be separated by sequence specific DNA affinity chromatography. These results confirm the observation that TFIIIC can be fractionated into two components, which are both required for transcription of VA I and tRNA genes in vitro. Thus in the mammalian reconstituted system, a minimum of three proteins, in addition to RNA polymerase III, are required for the transcription of the VA and tRNA genes in vitro. The DNA binding component, TFIIIC2, binds specifically to the 3' segment of the internal promoter (the B block), demonstrated by its ability to protect this region from digestion by DNase I. TFIIIC2 is the limiting, titratable component in the phosphocellulose C fraction required for the formation of a stable pre-initiation complex on the VAI RNA gene in vitro, as demonstrated with a template competition and rescue assay. Images PMID:3697084

  4. Purification of infective bluetongue virus particles by immuno-affinity chromatography using anti-core antibody.

    PubMed

    Chand, Karam; Biswas, Sanchay K; Mondal, Bimalendu

    2016-03-01

    An immuno-affinity chromatography technique for purification of infective bluetongue virus (BTV) has been descried using anti-core antibodies. BTV anti-core antibodies (prepared in guinea pig) were mixed with cell culture-grown BTV-1 and then the mixture was added to the cyanogens bromide-activated protein-A Sepharose column. Protein A binds to the antibody which in turn binds to the antigen (i.e. BTV). After thorough washing, antigen-antibody and antibody-protein A couplings were dissociated with 4M MgCl2, pH6.5. Antibody molecules were removed by dialysis and virus particles were concentrated by spin column ultrafiltration. Dialyzed and concentrated material was tested positive for BTV antigen by a sandwich ELISA and the infectivity of the chromatography-purified virus was demonstrated in cell culture. This method was applied for selective capture of BTV from a mixture of other viruses. As group-specific antibodies (against BTV core) were used to capture the virus, it is expected that virus of all BTV serotypes could be purified by this method. This method will be helpful for selective capture and enrichment of BTV from concurrently infected blood or tissue samples for efficient isolation in cell culture. Further, this method can be used for small scale purification of BTV avoiding ultracentrifugation. PMID:26925450

  5. Characterization of Extracellular Proteins in Tomato Fruit using Lectin Affinity Chromatography and LC-MALDI-MS/MS analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The large-scale isolation and analysis of glycoproteins by lectin affinity chromatography coupled with mass spectrometry has become a powerful tool to monitor changes in the “glycoproteome” of mammalian cells. Thus far, however, this approach has not been used extensively for the analysis of plant g...

  6. Evaluation and optimization of the metal-binding properties of a complex ligand for immobilized metal affinity chromatography.

    PubMed

    Chen, Bin; Li, Rong; Li, Shiyu; Chen, Xiaoli; Yang, Kaidi; Chen, Guoliang; Ma, Xiaoxun

    2016-02-01

    The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu(2+) to l-glutamic acid were measured, the metal ion-binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal-binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum-binding conditions of Cu(2+) on the l-glutamic acid-silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal-binding behaviors. PMID:26632098

  7. PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ACID PHOSPHATASE FROM SPIRODELA OLIGORRHIZA AND ITS AFFINITY FOR SELECTED ORGANOPHOSPHATE PESTICIDES

    EPA Science Inventory

    An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography (FPLC) and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. SDS-PAGE electrophoresis of the pure acid phosphatase ...

  8. Molecular weight abnormalities of the CTCF transcription factor: CTCF migrates aberrantly in SDS-PAGE and the size of the expressed protein is affected by the UTRs and sequences within the coding region of the CTCF gene.

    PubMed Central

    Klenova, E M; Nicolas, R H; U, S; Carne, A F; Lee, R E; Lobanenkov, V V; Goodwin, G H

    1997-01-01

    CTCF belongs to the Zn finger transcription factors family and binds to the promoter region of c-myc. CTCF is highly conserved between species, ubiquitous and localised in nuclei. The endogenous CTCF migrates as a 130 kDa (CTCF-130) protein on SDS-PAGE, however, the open reading frame (ORF) of the CTCF cDNA encodes only a 82 kDa protein (CTCF-82). In the present study we investigate this phenomenon and show with mass-spectra analysis that this occurs due to aberrant mobility of the CTCF protein. Another paradox is that our original cDNA, composed of the ORF and 3'-untranslated region (3'-UTR), produces a protein with the apparent molecular weight of 70 kDa (CTCF-70). This paradox has been found to be an effect of the UTRs and sequences within the coding region of the CTCF gene resulting in C-terminal truncation of CTCF-130. The potential attenuator has been identified and point-mutated. This restored the electrophoretic mobility of the CTCF protein to 130 kDa. CTCF-70, the aberrantly migrating CTCF N-terminus per se, is also detected in some cell types and therefore may have some biological implications. In particular, CTCF-70 interferes with CTCF-130 normal function, enhancing transactivation induced by CTCF-130 in COS6 cells. The mechanism of CTCF-70 action and other possible functions of CTCF-70 are discussed. PMID:9016583

  9. Comparing multistep immobilized metal affinity chromatography and multistep TiO2 methods for phosphopeptide enrichment.

    PubMed

    Yue, Xiaoshan; Schunter, Alissa; Hummon, Amanda B

    2015-09-01

    Phosphopeptide enrichment from complicated peptide mixtures is an essential step for mass spectrometry-based phosphoproteomic studies to reduce sample complexity and ionization suppression effects. Typical methods for enriching phosphopeptides include immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) beads, which have selective affinity and can interact with phosphopeptides. In this study, the IMAC enrichment method was compared with the TiO2 enrichment method, using a multistep enrichment strategy from whole cell lysate, to evaluate their abilities to enrich for different types of phosphopeptides. The peptide-to-beads ratios were optimized for both IMAC and TiO2 beads. Both IMAC and TiO2 enrichments were performed for three rounds to enable the maximum extraction of phosphopeptides from the whole cell lysates. The phosphopeptides that are unique to IMAC enrichment, unique to TiO2 enrichment, and identified with both IMAC and TiO2 enrichment were analyzed for their characteristics. Both IMAC and TiO2 enriched similar amounts of phosphopeptides with comparable enrichment efficiency. However, phosphopeptides that are unique to IMAC enrichment showed a higher percentage of multiphosphopeptides as well as a higher percentage of longer, basic, and hydrophilic phosphopeptides. Also, the IMAC and TiO2 procedures clearly enriched phosphopeptides with different motifs. Finally, further enriching with two rounds of TiO2 from the supernatant after IMAC enrichment or further enriching with two rounds of IMAC from the supernatant TiO2 enrichment does not fully recover the phosphopeptides that are not identified with the corresponding multistep enrichment. PMID:26237447

  10. Preparation of high capacity affinity adsorbents using new hydrazino-carriers and their use for low and high performance affinity chromatography of lectins.

    PubMed

    Ito, Y; Yamasaki, Y; Seno, N; Matsumoto, I

    1986-04-01

    Two kinds of carriers with high concentrations of hydrazino groups were prepared by simple and convenient procedures. Hydrazino-carriers (I) and (II) were obtained on incubation of epoxy-activated carriers with hydrazine hydrate and adipic acid dihydrazide, respectively. Disaccharides were coupled to the hydrazino carriers through reductive amination in the presence of sodium cyanoborohydride. The reaction time was much shorter (24 h) than that in the case of the method involving amino-Sepharose 6B (800 h) [Matsumoto, I., Kitagaki, H., Akai, Y., Ito, Y., & Seno, N. (1981) Anal. Biochem. 116, 103-110]. The glycamyl-Sepharose thus obtained showed high adsorption capacities for lectins. Glycamyl-TSKgel G3000 PW obtained by the same method with TSKgel G3000 PW, which is a hydrophobic vinyl polymer matrix for high performance gel permeation liquid chromatography, could be successfully used for the high performance liquid affinity chromatography of lectins. N-Acetylglutamic acid was coupled to hydrazino-Sepharose 4B (I) in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The adsorbent obtained was used for the affinity chromatography of Japanese horseshoe crab lectin. PMID:3711062

  11. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    PubMed

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

  12. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography

    PubMed Central

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases. PMID:26641240

  13. BIOINTERACTION ANALYSIS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: KINETIC STUDIES OF IMMOBILIZED ANTIBODIES

    PubMed Central

    Nelson, Mary Anne; Moser, Annette; Hage, David S.

    2009-01-01

    A system based on high-performance affinity chromatography was developed for characterizing the binding, elution and regeneration kinetics of immobilized antibodies and immunoaffinity supports. This information was provided by using a combination of frontal analysis, split-peak analysis and peak decay analysis to determine the rate constants for antibody-antigen interactions under typical sample application and elution conditions. This technique was tested using immunoaffinity supports that contained monoclonal antibodies for 2,4-dichlorophenoxyacetic acid (2,4-D). Association equilibrium constants measured by frontal analysis for 2,4-D and related compounds with the immobilized antibodies were 1.7–12 × 106 M−1 at pH 7.0 and 25°C. Split-peak analysis gave association rate constants of 1.4–12 × 105 M−1s−1 and calculated dissociation rate constants of 0.01–0.4 s−1 under the application conditions. Elution at pH 2.5 for the analytes from the antibodies was examined by peak decay analysis and gave dissociation rate constants of 0.056–0.17 s−1. A comparison of frontal analysis results after various periods of column regeneration allowed the rate of antibody regeneration to be examined, with the results giving a first-order regeneration rate constant of 2.4 × 10−4 s−1. This combined approach and the information it provides should be useful in the design and optimization of immunoaffinity chromatography and other analytical methods that employ immobilized antibodies. The methods described are not limited to the particular analytes and antibodies employed in this study but should be useful in characterizing other targets, ligands and supports. PMID:19394281

  14. Ligand affinity chromatography, an indispensable method for the purification of soluble cytokine receptors and binding proteins.

    PubMed

    Novick, Daniela; Rubinstein, Menachem

    2012-01-01

    Ligand affinity chromatography separation is based on unique interaction between the target analyte and a ligand, which is coupled covalently to a resin. It is a simple, rapid, selective, and efficient purification procedure of proteins providing tens of thousands fold purification in one step. The biological activity of the isolated proteins is retained in most cases thus function is revealed concomitantly with the isolation. Prior to the completion of the genome project this method facilitated rapid and reliable cloning of the corresponding gene. Upon completion of this project, a partial protein sequence is enough for retrieving its complete mRNA and hence its complete protein sequence. This method is indispensable for the isolation of both expected (e.g. receptors) but mainly unexpected, unpredicted and very much surprising binding proteins. No other approach would yield the latter. This chapter provides examples for both the expected target proteins, isolated from rich sources of human proteins, as well as the unexpected binding proteins, found by serendipity. PMID:22131033

  15. Immobilized metal affinity chromatography without chelating ligands: purification of soybean trypsin inhibitor on zinc alginate beads.

    PubMed

    Gupta, Munishwar N; Jain, Sulakshana; Roy, Ipsita

    2002-01-01

    Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu(2+), Zn(2+), and Ni(2+), which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn(2+) directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL(-1), as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity. PMID:11822903

  16. Affinity chromatography of Drosophila melanogaster ribosomal proteins to 5S rRNA.

    PubMed

    Stark, B C; Chooi, W Y

    1985-02-20

    The binding of Drosophila melanogaster ribosomal proteins to D. melanogaster 5S rRNA was studied using affinity chromatography of total ribosomal proteins (TP80) on 5S rRNA linked via adipic acid dihydrazide to Sepharose 4B. Ribosomal proteins which bound 5S rRNA at 0.3 M potassium chloride and were eluted at 1 M potassium chloride were identified as proteins 1, L4, 2/3, L14/L16, and S1, S2, S3, S4, S5, by two-dimensional polyacrylamide gel electrophoresis. Using poly A-Sepharose 4B columns as a model of non-specific binding, we found that a subset of TP80 proteins is also bound. This subset, while containing some of the proteins bound by 5S rRNA columns, was distinctly different from the latter subset, indicating that the binding to 5S rRNA was specific for that RNA species. PMID:3923010

  17. CHARACTERIZATION OF DRUG-PROTEIN INTERACTIONS IN BLOOD USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Hage, David S.; Jackson, Abby; Sobansky, Matt; Schiel, John E.; Yoo, Michelle J.; Joseph, K. S.

    2009-01-01

    The binding of drugs with proteins in blood, serum or plasma is an important process in determining the activity, distribution, rate of excretion, and toxicity of drugs in the body. High-performance affinity chromatography (HPAC) has received a great deal of interest as a means for studying these interactions. This review examines the various techniques that have been used in HPAC to examine drug-protein binding and discusses the types of information that can be obtained through this approach. A comparison of these techniques with traditional methods for binding studies (e.g., equilibrium dialysis and ultrafiltration) will also be presented. The use of HPAC with specific serum proteins and binding agents will then be discussed, including human serum albumin and α1-acid glycoprotein. Several examples from the literature are provided to illustrate the applications of such research. Recent developments in this field are also described, such as the use of improved immobilization techniques, new data analysis methods, techniques for working for directly with complex biological samples, and work with immobilized lipoproteins. The relative advantages and limitations of the methods that are described will be considered and the possible use of these techniques in the high-throughput screening or characterization of drug-protein binding will be discussed. PMID:19278006

  18. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography

    PubMed Central

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S.

    2015-01-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  19. MEASUREMENT OF DRUG-PROTEIN DISSOCIATION RATES BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PEAK PROFILING

    PubMed Central

    Schiel, John E.; Ohnmacht, Corey M.; Hage, David S.

    2012-01-01

    The rate at which a drug or other small solute interacts with a protein is important in understanding the biological and pharmacokinetic behavior of these agents. One approach that has been developed for examining these rates involves the use of high-performance affinity chromatography (HPAC) and estimates of band-broadening through peak profiling. Previous work with this method has been based on a comparison of the statistical moments for a retained analyte versus non-retained species at a single, high flow rate to obtain information on stationary phase mass transfer. In this study an alternative approach was created that allows a broad range of flow rates to be used for examining solute-protein dissociation rates. Chromatographic theory was employed to derive equations that could be used with this approach on a single column, as well as with multiple columns to evaluate and correct for the impact of stagnant mobile phase mass transfer. The interaction of L-tryptophan with human serum albumin was used as a model system to test this method. A dissociation rate constant of 2.7 (± 0.2) s−1 was obtained by this approach at pH 7.4 and 37°C, which was in good agreement with previous values determined by other methods. The techniques described in this report can be applied to other biomolecular systems and should be valuable for the determination of drug-protein dissociation rates. PMID:19422253

  20. Dynamic affinity chromatography in the separation of sulfated lignins binding to thrombin

    PubMed Central

    Liang, Aiye; Thakkar, Jay N.; Hindle, Michael; Desai, Umesh R.

    2013-01-01

    Sulfated low molecular weight lignins (LMWLs), a mixture of chemo-enzymatically prepared oligomers, have been found to be potent antagonists of coagulation. However, structures that induce anticoagulation remain unidentified. The highly polar sulfate groups on these molecules and the thousands of different structures present in these mixtures make traditional chromatographic resolution of sulfated LMWLs difficult. We performed dynamic thrombin affinity chromatography monitored using chromogenic substrate hydrolysis assay to isolate sulfated LMWL fractions that differed significantly in their biophysical and biochemical properties. Three fractions, I35, I55 and Peak II, were isolated from the starting complex mixture. Independent plasma clotting assays suggested that I35 possessed good anticoagulation potential (APTT = 4.2 μM; PT = 6.8 μM), while I55 and Peak II were approximately 10- and 100-fold less potent. The ESI-MS spectrum of this oligomeric fraction showed multiple peaks at 684.8, 610.6, 557.4, 541.4, 536.5, and 519.4 m/z, which most probably arise from variably functionalized (β-O4—β-β-linked trimers and/or a β-O4—β-O4-linked dimers. The first direct observation of these structures in sulfated LMWLs will greatly assist in the discovery of more potent sulfated LMWL-based anticoagulants. PMID:23122400

  1. CHARACTERIZATION OF DRUG INTERACTIONS WITH SERUM PROTEINS BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe; Barnaby, Omar; Jackson, Abby; Yoo, Michelle J.; Papastavros, Efthimia; Pfaunmiller, Erika; Sobansky, Matt; Tong, Zenghan

    2011-01-01

    The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding. PMID:21395530

  2. Analysis of Lidocaine Interactions with Serum Proteins Using High-Performance Affinity Chromatography

    PubMed Central

    Soman, Sony; Yoo, Michelle J.; Jang, Yoon Jeong; Hage, David S.

    2010-01-01

    High-performance affinity chromatography was used to study binding by the drug lidocaine to human serum albumin (HSA) and α1–acid glycoprotein (AGP). AGP had strong binding to lidocaine, with an association equilibrium constant (Ka) of 1.1-1.7 × 105 M-1 at 37 °C and pH 7.4. Lidocaine had weak-to-moderate binding to HSA, with a Ka in the range of 103 to 104 M-1. Competitive experiments with site selective probes showed that lidocaine was interacting with Sudlow site II of HSA and the propranolol site of AGP. These results agree with previous observations in the literature and provide a better quantitative understanding of how lidocaine binds to these serum proteins and is transported in the circulation. This study also demonstrates how HPAC can be used to examine the binding of a drug with multiple serum proteins and provide detailed information on the interaction sites and equilibrium constants that are involved in such processes. PMID:20138813

  3. Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

    PubMed Central

    Choi, Ryan; Kelley, Angela; Leibly, David; Nakazawa Hewitt, Stephen; Napuli, Alberto; Van Voorhis, Wesley

    2011-01-01

    The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure. PMID:21904040

  4. Characterization of glycoproteins in pancreatic cyst fluid using a high performance multiple lectin affinity chromatography platform

    PubMed Central

    Gbormittah, Francisca Owusu; Haab, Brian B.; Partyka, Katie; Garcia-Ott, Carolina; Hancapie, Marina; Hancock, William S.

    2014-01-01

    Currently, pancreatic cancer is the fourth cause of cancer death. In 2013, it is estimated that approximately 38,460 people will die of pancreatic cancer. Early detection of malignant cyst (pancreatic cancer precursor) is necessary to help prevent late diagnosis of the tumor. In this study, we characterized glycoproteins and non-glycoproteins on pooled mucinous (n=10) and non-mucinous (n=10) pancreatic cyst fluid to identify ‘proteins of interest’ to differentiate between mucinous cyst from non-mucinous cyst and investigate these proteins as potential biomarker targets. An automated multi-lectin affinity chromatography (M-LAC) platform was utilized for glycoprotein enrichment followed by nano-LC-MS/MS analysis. Spectral count quantitation allowed for the identification of proteins with significant differential levels in mucinous cysts from non-mucinous cysts of which one protein (periostin) was confirmed via immunoblotting. To exhaustively evaluate differentially expressed proteins, we used a number of proteomic tools including; gene ontology classification, pathway and network analysis, Novoseek data mining and chromosome gene mapping. Utilization of complementary proteomic tools, revealed that several of the proteins such as mucin 6 (MUC6), bile salt-activated lipase (CEL) and pyruvate kinase lysozyme M1/M2 with significant differential expression have strong association with pancreatic cancer. Further, chromosome gene mapping demonstrated co-expressions and co-localization of some proteins of interest including 14-3-3 protein epsilon (YWHAE), pigment epithelium derived factor (SERPINF1) and oncogene p53. PMID:24303806

  5. Advance chromatin extraction improves capture performance of protein A affinity chromatography.

    PubMed

    Nian, Rui; Zhang, Wei; Tan, Lihan; Lee, Jeremy; Bi, Xeuzhi; Yang, Yuansheng; Gan, Hui Theng; Gagnon, Pete

    2016-01-29

    Practical effects of advance chromatin removal on performance of protein A affinity chromatography were evaluated using a caprylic acid-allantoin-based extraction method. Lacking this treatment, the practice of increasing loading residence time to increase capacity was shown to increase host protein contamination of the eluted IgG. Advance chromatin extraction suspended that compromise. Protein A ligand leakage from columns loaded with chromatin-extracted harvest was half the level observed on protein A columns loaded with non-extracted harvest. Columns loaded with chromatin-extracted harvest were cleaned more effectively by 50-100mM NaOH than columns loaded with non-extracted harvest that were cleaned with 250-500mM NaOH. Two protein A media with IgG capacities in excess of 50g/L were loaded with chromatin-extracted harvest, washed with 2.0M NaCl before elution, and the eluted IgG fraction titrated to pH 5.5 before microfiltration. Host protein contamination in the filtrate was reduced to <1ppm, DNA to <1ppb, protein A leakage to 0.5ppm, and aggregates to 1.0%. Caprylic acid and allantoin were both reduced below 5ppm. Step recovery of IgG was 99.4%. Addition of a single polishing step reduced residual protein A beneath the level of detection and aggregates to <0.1%. Overall process recovery including chromatin extraction was 90%. PMID:26774119

  6. Analysis of Drug Interactions with Lipoproteins by High-Performance Affinity Chromatography

    PubMed Central

    Sobansky, Matthew R.; Hage, David S.

    2013-01-01

    Lipoproteins such as high-density lipoprotein (HDL) and low-density lipoprotein (LDL) are known to interact with drugs and other solutes in blood. These interactions have been examined in the past by methods such as equilibrium dialysis and capillary electrophoresis. This chapter describes an alternative approach that has recently been developed for examining these interactions by using high-performance affinity chromatography. In this method, lipoproteins are covalently immobilized to a solid support and used within a column as a stationary phase for binding studies. This approach allows the same lipoprotein preparation to be used for a large number of binding studies, leading to precise estimates of binding parameters. This chapter will discuss how this technique can be applied to the identification of interaction models and be used to differentiate between systems that have interactions based on partitioning, adsorption or mixed-mode interactions. It is also shown how this approach can then be used for the measurement of binding parameters for HDL and LDL with drugs. Examples of these studies are provided, with particular attention being given to the use of frontal analysis to examine the interactions of R- and S-propranolol with HDL and LDL. The advantages and possible limitations of this method are described. The extension of this approach to other types of drug-lipoprotein interactions is also considered. PMID:25392741

  7. Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography.

    PubMed

    Matsuda, Ryan; Kye, So-Hwang; Anguizola, Jeanethe; Hage, David S

    2014-08-01

    Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered. PMID:26526139

  8. The Lectin Frontier Database (LfDB), and data generation based on frontal affinity chromatography.

    PubMed

    Hirabayashi, Jun; Tateno, Hiroaki; Shikanai, Toshihide; Aoki-Kinoshita, Kiyoko F; Narimatsu, Hisashi

    2015-01-01

    Lectins are a large group of carbohydrate-binding proteins, having been shown to comprise at least 48 protein scaffolds or protein family entries. They occur ubiquitously in living organisms-from humans to microorganisms, including viruses-and while their functions are yet to be fully elucidated, their main underlying actions are thought to mediate cell-cell and cell-glycoconjugate interactions, which play important roles in an extensive range of biological processes. The basic feature of each lectin's function resides in its specific sugar-binding properties. In this regard, it is beneficial for researchers to have access to fundamental information about the detailed oligosaccharide specificities of diverse lectins. In this review, the authors describe a publicly available lectin database named "Lectin frontier DataBase (LfDB)", which undertakes the continuous publication and updating of comprehensive data for lectin-standard oligosaccharide interactions in terms of dissociation constants (Kd's). For Kd determination, an advanced system of frontal affinity chromatography (FAC) is used, with which quantitative datasets of interactions between immobilized lectins and >100 fluorescently labeled standard glycans have been generated. The FAC system is unique in its clear principle, simple procedure and high sensitivity, with an increasing number (>67) of associated publications that attest to its reliability. Thus, LfDB, is expected to play an essential role in lectin research, not only in basic but also in applied fields of glycoscience. PMID:25580689

  9. Analysis of free drug fractions in human serum by ultrafast affinity extraction and two-dimensional affinity chromatography.

    PubMed

    Zheng, Xiwei; Podariu, Maria; Matsuda, Ryan; Hage, David S

    2016-01-01

    Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research. PMID:26462924

  10. Affinity chromatography of trypsin and related enzymes. III. Purification of Streptomyces griseus trypsin using an affinity adsorbent containing a tryptic digest of protamine as a ligand.

    PubMed

    Yokosawa, H; Hanba, T; Ishii, S

    1976-04-01

    A new, simple method has been developed for the purification of Streptomyces griseus trypsin [EC 3.4.21.4] from Pronase. Only a single operation of affinity chromatography on an agarose derivative, which was easily prepared by coupling a tryptic digest of salmine to cyanogen bromide-activated Sepharose 4B, was required. A high degree of homogeneity was demonstrated for the purified enzyme by disc electrophoresis, SDS-polyacrylamide gel electrophoresis and gel filtration, as well as by active-site titration. The behavior of a carboxypeptides B [EC 3.4.12.3]-like enzyme present in Pronase is also discussed. PMID:819428

  11. A novel matrix derivatized from hydrophilic gigaporous polystyrene-based microspheres for high-speed immobilized-metal affinity chromatography.

    PubMed

    Qu, Jian-Bo; Huang, Yong-Dong; Jing, Guang-Lun; Liu, Jian-Guo; Zhou, Wei-Qing; Zhu, Hu; Lu, Jian-Ren

    2011-05-01

    Agarose coated gigaporous polystyrene microspheres were evaluated as a novel matrix for immobilized-metal affinity chromatography (IMAC). With four steps, nickel ions were successfully immobilized on the microspheres. The gigaporous structure and chromatographic properties of IMAC medium were characterized. A column packed with the matrix showed low column backpressure and high column efficiency at high flow velocity. Furthermore, this matrix was used for purifying superoxide dismutase (SOD), which was expressed in Escherichia coli (E. coli) in submerged fermentation, on an Äkta purifier 100 system under different flow velocities. The purity of the SOD from this one-step purification was 79% and the recovery yield was about 89.6% under the superficial flow velocity of 3251 cm/h. In conclusion, all the results suggested that the gigaporous matrix has considerable advantages for high-speed immobilized-metal affinity chromatography. PMID:21454141

  12. Identification of high-affinity calmodulin-binding proteins in rat liver

    SciTech Connect

    Hanley, R.M.; Dedman, J.R.; Shenolikar, S.

    1987-03-01

    The Ca/sup 2 +/-dependent binding of (/sup 125/I) calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or acceptor proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised > 80% of the Ca/sup 2 +/-dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding subunit of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca/sup 2 +/ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver.

  13. Optimization of human serum albumin monoliths for chiral separations and high-performance affinity chromatography.

    PubMed

    Pfaunmiller, Erika L; Hartmann, Mahli; Dupper, Courtney M; Soman, Sony; Hage, David S

    2012-12-21

    Various organic-based monoliths were prepared and optimized for immobilization of the protein human serum albumin (HSA) as a binding agent for chiral separations and high-performance affinity chromatography. These monoliths contained co-polymers based on glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) or GMA and trimethylolpropane trimethacrylate (TRIM). A mixture of cyclohexanol and 1-dodecanol was used as the porogen, with the ratio of these solvents being varied along with the polymerization temperature to generate a library of monoliths. These monoliths were used with both the Schiff base and epoxy immobilization methods and measured for their final content of HSA. Monoliths showing the highest protein content were further evaluated in chromatographic studies using R/S-warfarin and d/l-tryptophan as model chiral solutes. A 2.6-2.7-fold increase in HSA content was obtained in the final monoliths when compared to similar HSA monoliths prepared according to the literature. The increased protein content made it possible for the new monoliths to provide higher retention and/or two-fold faster separations for the tested solutes when using 4.6mm i.d.× 50 mm columns. These monoliths were also used to create 4.6mm i.d.× 10 mm HSA microcolumns that could separate the same chiral solutes in only 1.5-6.0 min. The approaches used in this study could be extended to the separation of other chiral solutes and to the optimization of organic monoliths for use with additional proteins as binding agents. PMID:23010249

  14. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

    PubMed Central

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-01-01

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL−1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL−1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

  15. Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

    PubMed Central

    Ruprecht, Benjamin; Koch, Heiner; Medard, Guillaume; Mundt, Max; Kuster, Bernhard; Lemeer, Simone

    2015-01-01

    Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO2, Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 μg to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median cv of 15%). The number of unique phosphopeptides could be increased to more than 14,000 when the IMAC eluate was subjected to a subsequent hydrophilic strong anion exchange separation. Fe-IMAC columns outperformed Ti-IMAC and TiO2 in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow-throughs showed that all materials largely bound the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution, and the stochastic nature of data-dependent acquisition in mass spectrometry are the causes of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis that can be applied to a wide range of biological systems. PMID

  16. Optimization of human serum albumin monoliths for chiral separations and high-performance affinity chromatography

    PubMed Central

    Pfaunmiller, Erika L.; Hartmann, Mahli; Dupper, Courtney M.; Soman, Sony; Hage, David S.

    2012-01-01

    Various organic-based monoliths were prepared and optimized for immobilization of the protein human serum albumin (HSA) as a binding agent for chiral separations and high-performance affinity chromatography. These monoliths contained co-polymers based on glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) or GMA and trimethylolpropane trimethacrylate (TRIM). A mixture of cyclohexanol and 1-dodecanol was used as the porogen, with the ratio of these solvents being varied along with the polymerization temperature to generate a library of monoliths. These monoliths were used with both the Schiff base and epoxy immobilization methods and measured for their final content of HSA. Monoliths showing the highest protein content were further evaluated in chromatographic studies using R/S-warfarin and d/l-tryptophan as model chiral solutes. A 2.6–2.7-fold increase in HSA content was obtained in the final monoliths when compared to similar HSA monoliths prepared according to the literature. The increased protein content made it possible for the new monoliths to provide higher retention and/or two-fold faster separations for the tested solutes when using 4.6 mm i.d. × 50 mm columns. These monoliths were also used to create 4.6 mm i.d. × 10 mm HSA microcolumns that could separate the same chiral solutes in only 1.5–6.0 min. The approaches used in this study could be extended to the separation of other chiral solutes and to the optimization of organic monoliths for use with additional proteins as binding agents. PMID:23010249

  17. Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography.

    PubMed

    Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

    2016-04-01

    A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL(-1), Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL(-1). The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

  18. Glycoproteomic analysis of embryonic stem cells: identification of potential glycobiomarkers using lectin affinity chromatography of glycopeptides

    PubMed Central

    Alvarez-Manilla, Gerardo; Warren, Nicole L.; Atwood, James; Orlando, Ron; Dalton, Stephen; Pierce, Michael

    2011-01-01

    Numerous studies have recently focused on the identification of specific glycan biomarkers; given the important roles that protein linked glycans play, for example, during development and disease progression. The identification of protein glycobiomarkers, which are part of a very complex proteome, has involved the use of fractionation techniques such as lectin affinity chromatography. In this study, the glycoproteomic characterization of pluripotent murine embryonic stem cells (ES) and from ES cells that were differentiated into embroid bodies (EB) was performed using immobilized Concanavalin A (ConA). This procedure allowed the isolation of glycopeptides that express biantennary and hybrid N-linked structures (ConA2 fraction) as well as high mannose glycans (ConA3 fraction), that were abundant in both ES and EB stages. A total of 293 unique N-linked glycopeptide sequences (from 180 glycoproteins) were identified in the combined data sets from ES and EB cells. Of these glycopeptides, a total of 119 sequences were identified exclusively in only one of the lectin bound fractions, (24 in the ES-ConA2, 15 in the ES-ConA3, 16 in the EB-ConA2 and 64 in the EB-ConA3). Results from this study allowed the identification of individual N-glycosylation sites of proteins that express specific glycan types. The absence of some of these lectin bound glycopeptides in a cell stage suggested that they were derived from proteins that were either expressed exclusively on a defined developmental stage, or were expressed in both cell stages but carried the lectin bound oligosaccharides in only one of them. Therefore, these lectin bound glycopeptides can be considered as stage specific glycobiomarkers. PMID:19545112

  19. Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5.8 S ribosomal ribonucleic acid.

    PubMed

    Ulbrich, N; Lin, A; Wool, I G

    1979-09-10

    The proteins that bind to rat liver 5.8 S ribosomal ribonucleic acid were identified by affinity chromatography. The nucleic acid was oxidized with periodate and coupled by its 3'-terminus to Sepharose 4B through and adipic acid dihydrazide spacer. The ribosomal proteins that associate with the immobilized 5.8 S rRNA were identified by polyacrylamide gel electrophoresiss: they were L19, L8, and L6 from the 60 S subunit; and S13 and S9 from the small subparticle. Small amounts of L14, L17', L18, L27/L27', and L35', and of S11, S15, S23/S24, and S26 also were bound to the affinity column, but whether they associate directly and specifically with 5.8 S rRNA is not known. Escherichia coli ribosomal proteins did not bind to the rat liver 5.8 S rRNA affinity column. PMID:468846

  20. Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography.

    PubMed

    O'Shaughnessy, Colette M; Micoli, Francesca; Gavini, Massimiliano; Goodall, Margaret; Cobbold, Mark; Saul, Allan; Maclennan, Calman A

    2013-01-31

    Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg-ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg-ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg-ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella

  1. Detection of human butyrylcholinesterase-nerve gas adducts by liquid chromatography-mass spectrometric analysis after in gel chymotryptic digestion.

    PubMed

    Tsuge, Kouichiro; Seto, Yasuo

    2006-06-21

    To verify the exposure to nerve gas, a method for detecting human butyrylcholinesterase (BuChE)-nerve gas adduct was developed using LC-electrospray mass spectrometry (ESI-MS). Purified human serum BuChE was incubated with sarin, soman or VX, and the adduct was purified by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and digested in gel by treatment with chymotrypsin. The resulting peptide mixture was subjected to LC-ESI-MS. From the chymotryptic digest of untreated human BuChE, one peak corresponding to the peptide fragment containing the active center serine residue was detected on the extracted ion chromatogram at m/z 948.5, and the sequence was ascertained to be "GESAGAASVSL" by MS/MS analysis. From the chymotryptic digest of the human BuChE-sarin adduct, a singly charged peptide peak was detected on the extracted ion chromatogram at m/z 1,069.5, and the sequence was ascertained to be "GEXAGAASVSL" by MS/MS analysis (X denotes isopropylmethylphosphonylated serine). The difference in molecular weight (120.0 Da) between the active center peptide fragments corresponding to the untreated BuChE and BuChE-sarin adduct was assumed to be derived from the addition of an isopropyl methylphosphonyl moiety to the serine residue. The formation of human BuChE adducts with soman, VX and an aged soman adduct was confirmed by detecting the respective active center peptide fragments using LC-ESI-MS. To apply the established method to an actual biological sample, human serum was incubated with VX, and the adduct was purified by procainamide affinity chromatography followed by SDS-PAGE. After chymotryptic in gel digestion, the ethylphosphonylated active center peptide fragment could be detected, and the structure of the residue was ascertained by LC-ESI-MS analysis. PMID:16569519

  2. Identification of Novel in vivo MAP Kinase Substrates in Arabidopsis thaliana Through Use of Tandem Metal Oxide Affinity Chromatography*

    PubMed Central

    Hoehenwarter, Wolfgang; Thomas, Martin; Nukarinen, Ella; Egelhofer, Volker; Röhrig, Horst; Weckwerth, Wolfram; Conrath, Uwe; Beckers, Gerold J. M.

    2013-01-01

    Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)3-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO2-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment. PMID:23172892

  3. Use of Tandem Affinity Chromatography for Purification of Cannabinoid Receptor CB2

    PubMed Central

    Locatelli-Hoops, Silvia C.; Yeliseev, Alexei A.

    2016-01-01

    Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor. PMID:24943318

  4. Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization, Part 1: Theory

    PubMed Central

    2015-01-01

    We present a novel technique that couples isotachophoresis (ITP) with affinity chromatography (AC) to achieve rapid, selective purification with high column utilization. ITP simultaneously preconcentrates an analyte and purifies it, based on differences in mobility of sample components, excluding species that may foul or compete with the target at the affinity substrate. ITP preconcentration accelerates the affinity reaction, reducing assay time, improving column utilization, and allowing for capture of targets with higher dissociation constants. Furthermore, ITP-AC separates the target and contaminants into nondiffusing zones, thus achieving high resolution in a short distance and time. We present an analytical model for spatiotemporal dynamics of ITP-AC. We identify and explore the effect of key process parameters, including target distribution width and height, ITP zone velocity, forward and reverse reaction constants, and probe concentration on necessary affinity region length, assay time, and capture efficiency. Our analytical approach shows collapse of these variables to three nondimensional parameters. The analysis yields simple analytical relations for capture length and capture time in relevant ITP-AC regimes, and it demonstrates how ITP greatly reduces assay time and improves column utilization. In the second part of this two-part series, we will present experimental validation of our model and demonstrate ITP-AC separation of the target from 10,000-fold more-abundant contaminants. PMID:24937679

  5. Direct capture of His₆-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

    PubMed

    Singh, Naveen Kumar; DSouza, Roy N; Bibi, Noor Shad; Fernández-Lahore, Marcelo

    2015-01-01

    Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels. PMID:25749956

  6. Use of differential dye-ligand chromatography with affinity elution for enzyme purification: 6-phosphogluconate dehydratase from Zymomonas mobilis.

    PubMed

    Scopes, R K; Griffiths-Smith, K

    1984-02-01

    Using differential dye-ligand chromatography and affinity elution with a substrate analog, 6-phosphogluconate dehydratase (EC 4.2.1.12) has been isolated from extracts of Zymomonas mobilis in a one-step procedure with 50% recovery. The specific activity of freshly isolated enzyme was 245 units mg-1. The enzyme contains iron, and it is rapidly inactivated in oxidizing conditions. It is inhibited by glycerophosphates, most strongly by the D-alpha-isomer which structurally corresponds to half of the substrate molecule. PMID:6326623

  7. Isolation and purification of cat albumin from cat serum by copper ion affinity chromatography: further analysis of its primary structure.

    PubMed

    Dandeu, J P; Rabillon, J; Guillaume, J L; Camoin, L; Lux, M; David, B

    1991-02-22

    Proteins, regardless of their origin, have to be highly purified, particularly from the immunochemical point of view, if they are to be used to study their allergenicity. It is shown that cat albumin, a highly potent allergen for cat-sensitive humans, can be isolated and purified from cat serum using immobilized metal ion affinity chromatography (copper ions) instead of a salting-out process or precipitation with alcohol, techniques generally used for the preparation of serum proteins. During the process described, immunoglobulins are concomitantly isolated in a relatively pure form. Cat albumin amino acid composition and sequence were analysed after an ultimate purification by ion-exchange chromatography. The highest homology (greater than 80%) was found with the rat serum albumin. PMID:2045457

  8. Detection and Identification of Heme c-Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography-Mass Spectrometry, and Database Searching

    SciTech Connect

    Merkley, Eric D.; Anderson, Brian J.; Park, Jea H.; Belchik, Sara M.; Shi, Liang; Monroe, Matthew E.; Smith, Richard D.; Lipton, Mary S.

    2012-12-07

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry-(LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed, or if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, a bacterial decaheme cytochrome, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded three- to six-fold more confident peptide-spectrum matches to heme-c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for four of the ten expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering nine out of ten sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 10-4 was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.

  9. A Highly Selective Hsp90 Affinity Chromatography Resin with a Cleavable Linker

    PubMed Central

    Hughes, Philip F; Barrott, Jared J; Carlson, David A; Loiselle, David R; Speer, Brittany L; Bodoor, Khaldon; Rund, Lauretta A; Haystead, Timothy A J

    2012-01-01

    Over 200 proteins have been identified that interact with the protein chaperone Hsp90, a recognized therapeutic target thought to participate in non-oncogene addiction in a variety of human cancers. However, defining Hsp90 clients is challenging because interactions between Hsp90 and its physiologically relevant targets involve low affinity binding and are thought to be transient. Using a chemo-proteomic strategy, we have developed a novel orthogonally cleavable Hsp90 affinity resin that allows purification of the native protein and is quite selective for Hsp90 over its immediate family members, GRP94 and TRAP 1. We show that the resin can be used under low stringency conditions for the rapid, unambiguous capture of native Hsp90 in complex with a native client. We also show that the choice of linker used to tether the ligand to the insoluble support can have a dramatic effect on the selectivity of the affinity media. PMID:22520629

  10. An optimized approach for enrichment of glycoproteins from cell culture lysates using native multi-lectin affinity chromatography.

    PubMed

    Lee, Ling Y; Hincapie, Marina; Packer, Nicolle; Baker, Mark S; Hancock, William S; Fanayan, Susan

    2012-09-01

    Lectins are capable of recognizing specific glycan structures and serve as invaluable tools for the separation of glycosylated proteins from nonglycosylated proteins in biological samples. We report on the optimization of native multi-lectin affinity chromatography, combining three lectins, namely, concanavalin A, jacalin, and wheat germ agglutinin for fractionation of cellular glycoproteins from MCF-7 breast cancer lysate. We evaluated several conditions for optimum recovery of total proteins and glycoproteins such as low pH and saccharide elution buffers, and the inclusion of detergents in binding and elution buffers. Optimum recovery was observed with overnight incubation of cell lysate with lectins at 4°C, and inclusion of detergent in binding and saccharide elution buffers. Total protein and bound recoveries were 80 and 9%, respectively. Importantly, we found that high saccharide strength elution buffers were not necessary to release bound glycoproteins. This study demonstrates that multi-lectin affinity chromatography can be extended to total cell lysate to investigate the cellular glycoproteome. PMID:22997032

  11. Column affinity chromatography for bound/free separation in ligand assays. I. Radioimmunoassay of choriomammotropin (human placental lactogen).

    PubMed

    Cornale, P; Bonazzi, M; Multinu, C; Romelli, P; Vancheri, L; Pennisi, F

    1981-06-01

    A method is described for separating antibody-bound from free fractions in ligand assays by column affinity chromatography, and its application to radioimmunoassay of choriomammotropin. In the method, 70 x 10 mm (i.d.) polypropylene columns containing about 150 mg of immunosorbent (goat anti-rabbit gamma-globulins covalently linked to Sepharose CL-4B) are used. Standards or unknowns, tracer and antiserum, pipetted into bottom-capped columns, are kept separated from the immunosorbent bed by a porous polyethylene disc and allowed to react for 15 min at room temperature. The reaction mixture is then allowed to pass through the columns by removing the bottom caps. Free antigen is eluted by washing the column, and discarded; antibody-bound fractions remain bound to the immunosorbent. The radioactivity in the columns is counted. The major advantages of the present technique, arising from the liquid-phase reaction combined with the solid-phase separation by column affinity chromatography, are the very low nonspecific binding (less than 1%), good sensitivity (0.02 mg/L), good precision (CV 3.4%), and simple and fast (30-min) assay. For 50 clinical samples so assayed (gamma) and compared with a polyethylene glycol precipitation technique (x), the regression equation was: y - 0.14 + 0.98x (r = 0.994). The assay method was clinical validated by 3493 determinations. PMID:7237770

  12. Enrichment and Analysis of Nonenzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron-Transfer Dissociation Mass Spectrometry

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Brock, Jonathan W.; Mottaz, Heather M.; Ames, Jennifer M.; Baynes, John; Smith, Richard D.; Metz, Thomas O.

    2007-06-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was observed that ETD fragmentation mode resulted in a significantly higher number of glycated peptide identifications (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing dual glycation enrichment on both the protein and peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS with ETD as the fragmentation mode is an efficient approach for analyses of glycated proteins and can have broad applications in studies of diabetes mellitus.

  13. Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to Multidimensional Protein Identification Technology

    PubMed Central

    Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E.; Yates, John R.

    2011-01-01

    In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases. PMID:21936497

  14. Sample displacement batch chromatography of proteins.

    PubMed

    Kotasinska, Marta; Richter, Verena; Kwiatkowski, Marcel; Schlüter, Hartmut

    2014-01-01

    In downstream processing large scale chromatography plays an important role. For its development screening experiments followed by pilot plant chromatography are mandatory steps. Here we describe fast, simple, and inexpensive methods for establishing a preparative chromatography for the separation of complex protein mixtures, based on sample displacement batch chromatography. The methods are demonstrated by anion-exchange chromatography of a human plasma protein fraction (Cohn IV-4), including the screening step and scaling up of the chromatography by a factor of 100. The results of the screening experiments and the preparative chromatography are monitored by SDS-PAGE electrophoresis. In summary we provide a protocol which should be easily adaptable for the chromatographic large scale purification of other proteins, in the laboratory as well as in industry for commercial manufacturing. For the latter these protocols cover the initial piloting steps for establishing a sample batch chromatography based on packed columns rather than batch chromatography. PMID:24648085

  15. Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

    PubMed Central

    Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

    2001-01-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  16. Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

    PubMed

    Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

    2001-02-01

    We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

  17. Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Morgan, Chad; Moir, Neil

    1996-11-01

    A rapid microscale procedure has been developed for the isolation and purification of yeast alcohol dehydrogenase. Glass beads are used for cytolysis, PEG precipitation for partial purification and a cibacron blue affinity column for the final step. A 27.5 fold purification can be achieved in 2 - 3 hours.

  18. Fundamental and practical studies on high-performance liquid affinity chromatography of biopolymers with novel stationary phases

    SciTech Connect

    Bacolod, M.D.

    1992-01-01

    Rigid microparticulate stationary phases having surface-bound metal chelating functions were developed and evaluated in high performance metal chelate affinity chromatography of proteins. Silica- and polystyrene-divinylbenzene-based metal chelate sorbents were produced in wide pore and in non-porous type of column packings. A major effort has been placed on development of non-porous highly crosslinked polystyrene-divinylbenzene (PSDVB). These PSDVB microparticles were produced by a two-step swelling polymerization, and exhibited excellent mechanical strength over a wide range of flow-rates and composition used in high performance liquid chromatography (HPLC). Simple and reproducible hydrophilic coatings were developed for the surface modification of hydrophobic PSDVB supports. A tetradentate metal chelating ligand, ethylenediamine-N, N[prime]-diacetic acid (EDDA), was covalently bound to the surface of the various supports. Sorbents having iminodiacetic acid (IDA) metal chelating functions were also evaluated. The hydrophilic character and surface coverage of various stationary phases were assessed chromatographically. Studies concerning the effects of eluent pH as well as the nature and concentration of salts on retention and selectivity with different metal chelate stationary phases having various immobilized metal ions were carried out. Elution schemes were developed for rapid separation of proteins in metal chelate affinity chromatography. EDDA stationary phases in metal forms can be viewed as complementary to IDA stationary phases since they afforded different selectivity and retentivity toward proteins. Hydrophilic PSDVB could be functionalized with IDA or EDDA metal chelating ligands or lectins. The non-porous metal chelate stationary phases afforded rapid separation of proteins by the development of multiple gradient systems, which permitted higher column peak capacity, enabling the separation of a greater number of proteins in a single chromatographic run.

  19. Octapeptide-based affinity chromatography of human immunoglobulin G: comparisons of three different ligands.

    PubMed

    Zhao, Wei-Wei; Liu, Fu-Feng; Shi, Qing-Hong; Sun, Yan

    2014-09-12

    In an earlier work, we have developed a biomimetic design strategy based on the human IgG (hIgG)-Protein A interactions and identified an affinity ligand for hIgG, FYWHCLDE, which ranked top one in a pool of 14 potential candidates. Herein, two more octapeptides, FYCHWALE and FYCHTIDE, were identified, and the binding and purification of hIgG on the affinity columns packed with the three octapeptide-modified Sepharose gels were extensively studied and compared to find more effective octapeptide-based affinity ligands. It was found that all the three ligands bound hIgG and Fc fragment but barely bound Fab fragment, and the binding to hIgG and Fc was mainly by electrostatic interactions. The optimum binding pH values for the three ligands were different from each other, but kept in the range of 5.0-6.0. Ligand binding competition revealed that the binding sites on hIgG for the three octapeptides were similar to those for Protein A. Adsorption isotherms revealed that hIgG binding capacity was in the range of 64-104mg/mL drained gel in the order of FYWHCLDE>FYCHWALE>FYCHTIDE. Then, purifications of hIgG and human monoclonal antibody from human serum and cell culture supernatant, respectively, were achieved with the three affinity columns at high purities and recovery yields. Finally, the molecular basis for the binding affinity of the peptides for the Fc fragment of hIgG was elucidated by molecular dynamics simulations. PMID:25064536

  20. Isolation of new pregnancy-associated glycoproteins from water buffalo (Bubalus bubalis) placenta by Vicia villosa affinity chromatography.

    PubMed

    Barbato, O; Sousa, N M; Klisch, K; Clerget, E; Debenedetti, A; Barile, V L; Malfatti, A; Beckers, J F

    2008-12-01

    The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8kDa and from 57.8 to 73.3kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta. PMID:18308351

  1. Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.

    PubMed

    Hellstern, Simon; Mutzel, Rupert

    2016-06-01

    We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ. Experiments with four chemically distinct calmodulin antagonists (R24571, Compound 48/80, melittin, and W7) showed that all of these substances inhibited the binding of purified TraC to calmodulin at effective concentrations comparable to those required for inhibiting in vitro binding of eukaryotic calmodulin-binding proteins. Three further bacterial proteins were identified as calmodulin-binding proteins: SecA, GlpD, and GlpC. We suggest that also these native bacterial proteins might be isolated by the unusual purification procedure including affinity chromatography on calmodulin Sepharose. Whether the identified proteins bind to, and are regulated by, putative bacterial calmodulin-like proteins in Escherichia coli remains to be established. PMID:26892535

  2. Coenzyme-like ligands for affinity isolation of cholesterol oxidase.

    PubMed

    Xin, Yu; Lu, Liushen; Wang, Qing; Zhang, Ling; Tong, Yanjun; Wang, Wu

    2016-05-15

    Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%. PMID:26856529

  3. Coupling isotachophoresis with affinity chromatography for rapid and selective purification with high column utilization, part 2: experimental study.

    PubMed

    Shkolnikov, Viktor; Santiago, Juan G

    2014-07-01

    We present an experimental study of coupling of isotachophoresis (ITP) and affinity chromatography (AC) to effect rapid, selective purification with high column utilization and high resolution. We provide a detailed protocol for performing ITP-AC and describe the design of a buffer system to perform sequence specific separation of nucleic acids. We describe the synthesis and functionalization of our affinity substrate, poly(glycidyl methacrylate-co-ethylene dimethacrylate) porous polymer monolith (GMA-EDMA PPM). This substrate allows easy immobilization of affinity probes, is nonsieving (even to macromolecules), and exhibits negligible nonspecific binding. We demonstrate ITP-AC with 25 nt, Cy5 labeled DNA target and a DNA probe and study the spatiotemporal dynamics using epifluorescence imaging. We make qualitative and quantitative comparisons between these data and the model presented in the first part of this two-paper series. We vary the target concentration from 1 pg μL(-1) to 100 pg μL(-1) and ITP velocity over the range of 10-50 μm s(-1), and thereby explore over 4 orders of magnitude of scaled target amount. We observe very good agreement between predictions and experimental data for the spatiotemporal behavior of the coupled ITP and affinity process, and for key figures of merit, including scaled capture length and maximum capture efficiency. Lastly, we demonstrate that the resolution of ITP-AC increases linearly with time and purify 25 nt target DNA from 10,000-fold higher abundance background (contaminating) genomic fish sperm DNA. We perform this capture from 200 μL of sample in under 1 mm column length and within <10 min. PMID:24937777

  4. Coupling Isotachophoresis with Affinity Chromatography for Rapid and Selective Purification with High Column Utilization, Part 2: Experimental Study

    PubMed Central

    2015-01-01

    We present an experimental study of coupling of isotachophoresis (ITP) and affinity chromatography (AC) to effect rapid, selective purification with high column utilization and high resolution. We provide a detailed protocol for performing ITP-AC and describe the design of a buffer system to perform sequence specific separation of nucleic acids. We describe the synthesis and functionalization of our affinity substrate, poly(glycidyl methacrylate-co-ethylene dimethacrylate) porous polymer monolith (GMA-EDMA PPM). This substrate allows easy immobilization of affinity probes, is nonsieving (even to macromolecules), and exhibits negligible nonspecific binding. We demonstrate ITP-AC with 25 nt, Cy5 labeled DNA target and a DNA probe and study the spatiotemporal dynamics using epifluorescence imaging. We make qualitative and quantitative comparisons between these data and the model presented in the first part of this two-paper series. We vary the target concentration from 1 pg μL–1 to 100 pg μL–1 and ITP velocity over the range of 10–50 μm s–1, and thereby explore over 4 orders of magnitude of scaled target amount. We observe very good agreement between predictions and experimental data for the spatiotemporal behavior of the coupled ITP and affinity process, and for key figures of merit, including scaled capture length and maximum capture efficiency. Lastly, we demonstrate that the resolution of ITP-AC increases linearly with time and purify 25 nt target DNA from 10 000-fold higher abundance background (contaminating) genomic fish sperm DNA. We perform this capture from 200 μL of sample in under 1 mm column length and within <10 min. PMID:24937777

  5. Applying DNA affinity chromatography to specifically screen for sucrose-related DNA-binding transcriptional regulators of Xanthomonas campestris.

    PubMed

    Leßmeier, Lennart; Alkhateeb, Rabeaa S; Schulte, Fabian; Steffens, Tim; Loka, Tobias Pascal; Pühler, Alfred; Niehaus, Karsten; Vorhölter, Frank-Jörg

    2016-08-20

    At a molecular level, the regulation of many important cellular processes is still obscure in xanthomonads, a bacterial group of outstanding relevance as world-wide plant pathogens and important for biotechnology as producers of the polysaccharide xanthan. Transcriptome analysis indicated a sucrose-dependent regulation of 18 genes in Xanthomonas campestris pv. campestris (Xcc) B100. The expression of 12 of these genes was clearly increased in the presence of sucrose. Only part of these genes was obviously involved in sucrose utilization. To identify regulatory proteins involved in transcriptional regulation, a DNA fragment-specific pull-down approach was established for Xcc. Putative promoter regions were identified and used to isolate DNA-binding proteins, which were separated by SDS PAGE and identified by MALDI-TOF mass spectrometry. This led to the identification of four transcriptional regulators, among them the global transcriptional regulator Clp and a previously identified regulator of sucrose utilization, SuxR, plus a third DNA-binding transcriptional regulator encoded by xcc-b100_2861 and recently shown to interact with a cyclic di-GMP-binding protein. The fourth regulatory protein was encoded by xcc-b100_2791. These results indicate DNA fragment-specific pull-down experiments as promising approaches to screen for specific DNA-binding regulatory proteins in Xcc. PMID:27060555

  6. Effect of cleaning agents and additives on Protein A ligand degradation and chromatography performance.

    PubMed

    Yang, Lihua; Harding, Jason D; Ivanov, Alexander V; Ramasubramanyan, Natarajan; Dong, Diane D

    2015-03-13

    Protein A chromatography, employing the recombinant Protein A ligand, is widely used as a capture step for antibody and Fc-fusion proteins manufacture. Protein A ligands in these matrices are susceptible to degradation/loss when exposed to cleaning agents such as sodium hydroxide, resulting in loss of capacity on reuse. In this study, MabSelect Protein A ligand and MabSelect SuRe Protein A ligand were chosen to evaluate the impact of alkaline cleaning solutions on the ligands and the packed columns. The Protein A ligands alone and the Protein A columns were incubated or cycled in different concentrations of sodium hydroxide solutions with and without additives, respectively. Ligand integrity (degradation) and ligand function (binding affinity) were studied using SDS-PAGE and customized Biacore technology, surface plasma resonance (SPR) and were successfully correlated with column performance measurement in terms of static binding capacity (SBC), dynamic binding capacity (DBC) and recovery as a function of exposure to cleaning agents with and without additives. The findings and the methodology presented in this study are not only able to determine appropriate cleaning conditions for Protein A chromatography, but also provided tools to enable systematic and rapid study of the cleaning solutions and conditions. PMID:25680549

  7. Affinity purification and characterization of a fibrinogen-binding protein complex which protects mice against lethal challenge with Streptococcus equi subsp. equi.

    PubMed

    Meehan, M; Nowlan, P; Owen, P

    1998-04-01

    Cell-wall-associated proteins from Streptococcus equi subsp. equi, the causative agent of strangles, were analysed with a view to identifying a potential protective antigen. Preparations of these proteins, isolated from mutanolysin extracts of cell walls, were shown to contain one major high-M(r) protein species (apparent M(r) 220,000 and 550,000 when analysed by SDS-PAGE and gel-filtration chromatography, respectively). The high-M(r) protein bound horse fibrinogen and was purified under non-denaturing conditions using fibrinogen affinity chromatography. The fibrinogen-binding protein (FgBP) reacted with serum taken from horses recovering from strangles and protected mice against lethal challenge from S. equi subsp. equi. The sequence of the corresponding gene (fbp) was determined and shown to encode a mature protein (M(r) 54,597) with predicted coiled-coil structure. An FgBP truncate, lacking the C-terminal cell wall/membrane anchor domain, was overexpressed in and purified from Escherichia coli and was shown to behave in an analogous fashion to the wild-type product in terms of M(r) estimation, fibrinogen binding and seroreactivity. PMID:9579073

  8. Purification of Hemoglobin from Red Blood Cells using Tangential Flow Filtration and Immobilized Metal Ion Affinity Chromatography

    PubMed Central

    Elmer, Jacob; Harris, David; Palmer, Andre F.

    2011-01-01

    Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are examined and compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter. PMID:21195679

  9. Evaluation of immobilized metal affinity chromatography kits for the purification of histidine-tagged recombinant CagA protein.

    PubMed

    Karakus, Cebrail; Uslu, Merve; Yazici, Duygu; Salih, Barik A

    2016-05-15

    Immobilized metal affinity chromatography (IMAC) technique is used for fast and reliable purification of histidine(His)-tagged recombinant proteins. The technique provides purification under native and denaturing conditions. The aim of this study is to evaluate three commercially available IMAC kits (Thermo Scientific, GE Healthcare and Qiagen) for the purification of a 6xHis-tagged recombinant CagA (cytotoxin-associated gene A) protein from IPTG-induced Escherichia coli BL21(DE3) culture. The kits were tested according to the manufacturer instructions and the protein was purified with only GE Healthcare and Qiagen kits under denaturing conditions. 1% (w/v) SDS was used as denaturing agent in PBS instead of extraction reagent of Thermo Scientific kit to lyse bacterial cells from 100ml culture. The 6xHis-tagged recombinant protein was purified by the three kits equally. PMID:26657801

  10. Imidazole-free purification of His3-tagged recombinant proteins using ssDNA aptamer-based affinity chromatography.

    PubMed

    Bartnicki, Filip; Kowalska, Ewa; Pels, Katarzyna; Strzalka, Wojciech

    2015-10-30

    Immobilized metal ion affinity chromatography (IMAC) is widely used for the purification of many different His6-tagged recombinant proteins. On the one hand, it is a powerful technique but on the other hand it has its disadvantages. In this report, we present the development of a unique ssDNA aptamer for the purification of His3-tagged recombinant proteins. Our study shows that stability of the His3-tag/H3T aptamer complex can be controlled by the sodium ion concentration. Based on this feature, we demonstrate that H3T aptamer resin was successfully employed for the purification of three out of four tested His3-tagged recombinant proteins from an E. coli total protein extract using imidazole-free buffers. Finally, we show that the purity of His3-tagged proteins is superior when purified with the help of the H3T aptamer in comparison with Ni-NTA resin. PMID:26427325

  11. Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography.

    PubMed

    Kim, Bong Gyun; Park, Hong Woo

    2015-01-01

    Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl2 . These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6-1.0 M arginine at pH 3.0-3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate. PMID:26363185

  12. Multi-Parameter Cell Affinity Chromatography: Separation and Analysis in a Single Microfluidic Channel

    PubMed Central

    Li, Peng; Gao, Yan; Pappas, Dimitri

    2012-01-01

    The ability to sort and capture more than one cell type from a complex sample will enable a wide variety of studies of cell proliferation, death, and the analysis of disease states. In this work, we integrated a pneumatic actuated control layer to an affinity separation layer to create different antibody coating regions on the same fluidic channel. The comparison of different antibody capture capabilities to the same cell line was demonstrated by flowing Ramos cells through anti-CD19 and anti-CD71 coated regions in the same channel, respectively. It was determined that cell capture density on anti-CD19 region was 2.44±0.13 times higher than on anti-CD71 coated region. This approach can be used to test different affinity molecules for selectivity and capture efficiency using a single cell line in one separation. Selective capture of Ramos and HuT 78 cells from a mixture was also demonstrated using two antibody regions in the same channel. Greater than 90% purity was obtained on both capture areas in both continuous flow and stop flow separation modes. A four-region antibody coated device was then fabricated to study the simultaneous, serial capture of three different cell lines. In this case the device showed effective capture of cells in a single separation channel, opening up the possibility of multiple cell sorting. Multi-parameter sequential blood sample analysis was also demonstrated with high capture specificity (>97% for both CD19+ and CD4+ leukocytes). The chip can also be used to selectively treat cells after affinity separation. PMID:22958145

  13. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    PubMed Central

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  14. Purification of the proprotein convertase furin by affinity chromatography based on PC-specific inhibitors

    PubMed Central

    Kuester, Miriam; Becker, Gero L.; Hardes, Kornelia; Lindberg, Iris; Steinmetzer, Torsten; Than, Manuel E.

    2013-01-01

    In eucaryotes, many secreted proteins and peptides are proteolytically excised from larger precursor proteins by a specific class of serine proteases, the proprotein/prohormone convertases (PCs). This cleavage is essential for substrate activation, making the PCs very interesting pharmacological targets in cancer and infectious disease research. Correspondingly, their structure, function and inhibition are intensely studied – studies that require the respective target proteins in large amounts and at high purity. Here we describe the development of a novel purification protocol of furin, the best-studied member of the PC family. We combined the heterologous expression of furin from CHO cells with a novel purification scheme employing an affinity step that efficiently extracts only active furin from the conditioned medium by using furin-specific inhibitor moieties as bait. Several potential affinity tags were synthesized and their binding to furin characterized. The best compound, Biotin-(Adoa)2-Arg-Pro-Arg-4-Amba coupled to streptavidin-Sepharose beads, was used in a three-step chromatographic protocol and routinely resulted in a high yield of a homogeneous furin preparation with a specific activity of ~60 units/mg protein. This purification and the general strategy can easily be adapted to the efficient purification of other PC family members. PMID:21875402

  15. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    SciTech Connect

    Evans, D.M.; Williams, K.P.; McGuinness, B.

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  16. The Plasma Membrane Ca(2+) ATPase: Purification by Calmodulin Affinity Chromatography, and Reconstitution of the Purified Protein.

    PubMed

    Niggli, Verena; Carafoli, Ernesto

    2016-01-01

    Plasma membrane Ca(2+) ATPases (PMCA pumps) are key regulators of cytosolic Ca(2+) in eukaryotes. They extrude Ca(2+) from the cytosol, using the energy of ATP hydrolysis and operate as Ca(2+)-H(+) exchangers. They are activated by the Ca(2+)-binding protein calmodulin, by acidic phospholipids and by other mechanisms, among them kinase-mediated phosphorylation. Isolation of the PMCA in pure and active form is essential for the analysis of its structure and function. In this chapter, the purification of the pump, as first achieved from erythrocyte plasma membranes by calmodulin-affinity chromatography, is described in detail. The reversible, high-affinity, Ca(2+)-dependent interaction of the pump with calmodulin is the basis of the procedure. Either phospholipids or glycerol have to be present in the isolation buffers to keep the pump active during the isolation procedure. After the isolation of the PMCA pump from human erythrocytes the pump was purified from other cell types, e.g., heart sarcolemma, plant microsomal fractions, and cells that express it ectopically. The reconstitution of the purified pump into phospholipid vesicles using the cholate dialysis method will also be described. It allows studies of transport mechanism and of regulation of pump activity. The purified pump can be stored in the reconstituted form for several days at 4 °C with little loss of activity, but it rapidly loses activity when stored in the detergent-solubilized form. PMID:26695022

  17. Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine.

    PubMed

    Kikot, Pamela; Polat, Aise; Achilli, Estefania; Fernandez Lahore, Marcelo; Grasselli, Mariano

    2014-11-01

    Fusion of peptide-based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram-range amounts of proteins. IMAC-Ni(II) columns have become the natural partners of 6xHis-tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His-tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur-containing molecules. In this work, we evaluated two different cysteine- and histidine-containing six amino acid tags linked to the N-terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine-containing tagged GFPs were able to bind to IMAC-Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC-Ni(II) system reaches less than 20% recovery of the cysteine-containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC-Pd(II) yields a recovery of 45% with a purification factor of 13. PMID:25277090

  18. NON-COMPETITIVE PEAK DECAY ANALYSIS OF DRUG-PROTEIN DISSOCIATION BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Chen, Jianzhong; Schiel, John E.; Hage, David S.

    2009-01-01

    The peak decay method is an affinity chromatographic technique that has been used to examine the dissociation of solutes from immobilized ligands in the presence of excess displacing agent. However, it can be difficult to find a displacing agent that does not interfere with detection of the eluting analyte. In this study, a non-competitive peak decay method was developed in which no displacing agent was required for analyte elution. This method was evaluated for the study of drug-protein interactions by using it along with high-performance affinity chromatography to measure the dissociation rate constants for R- and S-warfarin from columns containing immobilized human serum albumin (HSA). Several factors were considered in the optimization of this method, including the amount of applied analyte, the column size, and the flow rate. The dissociation rate constants for R- and S-warfarin from HSA were measured at several temperatures by this approach, giving values of 0.56 (± 0.01) and 0.66 (± 0.01) s−1 at pH 7.4 and 37°C. These results were in good agreement with previous values obtained by other methods. This approach is not limited to warfarin and HSA but could be employed in studying additional drug-protein interactions or other systems with weak-to-moderate binding. PMID:19472288

  19. Analysis of glipizide binding to normal and glycated human serum albumin by high-performance affinity chromatography.

    PubMed

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-07-01

    In diabetes, the elevated levels of glucose in the bloodstream can result in the nonenzymatic glycation of proteins such as human serum albumin (HSA). This type of modification has been shown to affect the interactions of some drugs with HSA, including several sulfonylurea drugs that are used to treat type II diabetes. This study used high-performance affinity chromatography (HPAC) to examine the interactions of glipizide (i.e., a second-generation sulfonylurea drug) with normal HSA or HSA that contained various levels of in vitro glycation. Frontal analysis indicated that glipizide was interacting with both normal and glycated HSA through two general groups of sites: a set of relatively strong interactions and a set of weaker interactions with average association equilibrium constants at pH 7.4 and 37 °C in the range of 2.4-6.0 × 10(5) and 1.7-3.7 × 10(4) M(-1), respectively. Zonal elution competition studies revealed that glipizide was interacting at both Sudlow sites I and II, which were estimated to have affinities of 3.2-3.9 × 10(5) and 1.1-1.4 × 10(4) M(-1). Allosteric effects were also noted to occur for this drug between the tamoxifen site and the binding of R-warfarin at Sudlow site I. Up to an 18% decrease in the affinity for glipizide was observed at Sudlow site I ongoing from normal HSA to glycated HSA, while up to a 27% increase was noted at Sudlow site II. This information should be useful in indicating how HPAC can be used to investigate other drugs that have complex interactions with proteins. These results should also be valuable in providing a better understanding of how glycation may affect drug-protein interactions and the serum transport of drugs such as glipizide during diabetes. PMID:25912461

  20. Identification of uranyl binding proteins from human kidney-2 cell extracts by immobilized uranyl affinity chromatography and mass spectrometry.

    PubMed

    Dedieu, Alain; Bérenguer, Frédéric; Basset, Christian; Prat, Odette; Quéméneur, Eric; Pible, Olivier; Vidaud, Claude

    2009-07-10

    To improve our knowledge on protein targets of uranyl ion (UO(2)(2+)), we set up a proteomic strategy based on immobilized metal-affinity chromatography (IMAC). The successful enrichment of UO(2)(2+)-interacting proteins from human kidney-2 (HK-2) soluble cell extracts was obtained using an ion-exchange chromatography followed by a dedicated IMAC process previously described and designed for the uranyl ion. By mass spectrometry analysis we identified 64 proteins displaying varied functions. The use of a computational screening algorithm along with the particular ligand-based properties of the UO(2)(2+) ion allowed the analysis and categorization of the protein collection. This profitable approach demonstrated that most of these proteins fulfill criteria which could rationalize their binding to the UO(2)(2+)-loaded phase. The obtained results enable us to focus on some targets for more in-depth studies and open new insights on its toxicity mechanisms at molecular level. PMID:19501829

  1. Chromatography.

    ERIC Educational Resources Information Center

    Brantley, L. Reed, Sr.; Demanche, Edna L.; Klemm, E. Barbara; Kyselka, Will; Phillips, Edwin A.; Pottenger, Francis M.; Yamamoto, Karen N.; Young, Donald B.

    This booklet presents some activities on chromatography. Directions for preparing leaf pigment extracts using alcohol are given, and paper chromatography and thin-layer chromatography are described as modifications of the basic principles of chromatography. (KHR)

  2. Interaction of L-glutamate oxidase with triazine dyes: selection of ligands for affinity chromatography.

    PubMed

    Katsos, N E; Labrou, N E; Clonis, Y D

    2004-08-01

    Glutamate oxidase (GOX, EC 1.4.3.11) from Streptomyces catalyses the oxidation of L-glutamate to alpha-ketoglutarate. Its kinetic constants for L-glutamate were measured equal to 2 mM for Km and 85.8 s(-1) for kcat. BLAST search and amino acid sequence alignments revealed low homology to other L-amino acid oxidases (18-38%). Threading methodology, homology modeling and CASTp analysis resulted in certain conclusions concerning the structure of catalytic alpha-subunit and led to the prediction of a binding pocket that provides favorable conditions of accommodating negatively charged aromatic ligands, such as sulphonated triazine dyes. Eleven commercial textile dyes and four biomimetic dyes or minodyes, bearing a ketocarboxylated-structure as their terminal biomimetic moiety, immobilized on cross-linked agarose gel. The resulted mini-library of affinity adsorbents was screened for binding and eluting L-glutamate oxidase activity. All but Cibacron Blue 3GA (CB3GA) affinity adsorbents were able to bind GOX at pH 5.6. One immobilized minodye-ligand, bearing as its terminal biomimetic moiety p-aminobenzyloxanylic acid (BM1), displayed the higher affinity for GOX. Kinetic inhibition studies showed that BM1 inhibits GOX in a non-competitive manner with a Ki of 10.5 microM, indicating that the dye-enzyme interaction does not involve the substrate-binding site. Adsorption equilibrium data, obtained from a batch system with BM1 adsorbent, corresponded well to the Freundlich isotherm with a rate constant k of 2.7 mg(1/2)ml(1/2)/g and Freundlich isotherm exponent n of 1. The interaction of GOX with the BM1 adsorbent was further studied with regards to adsorption and elution conditions. The results obtained were exploited in the development of a facile purification protocol for GOX, which led to 335-fold purification in a single step with high enzyme recovery (95%). The present purification procedure is the most efficient reported so far for L-glutamate oxidase. PMID:15203041

  3. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  4. Polymeric Cryogel-Based Boronate Affinity Chromatography for Separation of Ribonucleic Acid from Bacterial Extracts.

    PubMed

    Shakya, Akhilesh Kumar; Srivastava, Akshay; Kumar, Ashok

    2015-01-01

    Three-dimensional monolithic columns are preferred stationary phase in column chromatography. Conventional columns based on silica or particles are efficient in bioseparation though associated with limitations of nonspecific interaction and uneven porosity that causes high mass transfer resistance for the movement of big molecules. Cryogels as a monolith column have shown promising application in bioseparation. Cryogels column can be synthesized in the form of a monolith at sub-zero temperature through gelation of pre-synthesized polymers or polymerization of monomers. Cryogels are macroporous and mechanically stable materials. They have open interconnected micron-sized pores with a wide range of porosity (10-200 μm). Current protocol demonstrated the ability of poly(hydroxymethyl methacrylate)-co-vinylphenyl boronic acid p(HEMA-co-VPBA) cryogel matrix for selective separation of RNA from the bacterial crude extract. PMID:26623972

  5. Integrated bioprocess for the production and purification of recombinant proteins by affinity chromatography in Escherichia coli.

    PubMed

    Beshay, Usama; Miksch, Gerhard; Friehs, Karl; Flaschel, Erwin

    2009-02-01

    In order to improve the effectiveness of the production of recombinant proteins in E. coli, integrated fermentation processes were developed. Therefore, expression vectors were constructed containing a strongly expressed gene for a beta-glucanase fused with a metal-chelating affinity tag and a leader peptide for directing the fusion protein into the periplasmic space. Its export into the medium was achieved by means of co-expression of a bacteriocin-release protein, the Kil protein from pColE1. Bioreactors were modified so that special devices containing metal chelate pentadentate chelator PDC resins were located within the bioreactor. Using the bioreactor with an internal device the Zn2+-PDC had a 4.3-fold higher binding capacity than metal-free PDC (12.3 and 2.6 kU ml(-1) PDC, respectively. Using the bioreactor with charged PDC in an external circuit revealed even higher beta-glucanase concentration (65.6 kU ml(-1)), i.e. 1.5-fold compared to the internal adsorbent system. PMID:18481103

  6. Rapid purification of cytosolic epoxide hydrolase from normal and clofibrate-treated animals by affinity chromatography.

    PubMed Central

    Prestwich, G D; Hammock, B D

    1985-01-01

    Epoxide hydrolase from liver cytosol (cEH) of both normal and clofibrate-treated mice can be bioselectively adsorbed onto an affinity column prepared from epoxy-activated Sepharose and 7-methoxycitronellyl thiol. The free ligand is a modest inhibitor of cEH (I50, approximately equal to 3 X 10(-4) M) and lacks the epoxide function necessary for it to be turned over as a substrate. This study demonstrates that a methoxy group can be used to mimic an oxirane in a vertebrate system. Bioselective elution of cEH can be accomplished with several chalcone oxides, which are selective potent inhibitors (I50, 1-50 X 10(-7) M), and activity can be recovered by dialysis. This procedure thus enhances the purification by offering independent opportunities for selective binding and selective elution. Conservatively, a 40%-80% recovery of partially inhibited enzyme activity can be achieved in 4-48 hr with a 30- to 90-fold purification. The purified cEH from clofibrate-induced animals was essentially homogeneous by NaDodSO4/PAGE and had an apparent subunit molecular weight of 58,000. The cEHs from normal and clofibrate-induced animals appeared identical by NaDodSO4/PAGE. Since the cEH activity in normal and clofibrate-treated animals is due to the same enzyme, the increase in cEH activity caused by selected hypolipidemic agents appears to be true induction. Images PMID:3856846

  7. Isolation of human beta-interferon receptor by wheat germ lectin affinity and immunosorbent column chromatographies

    SciTech Connect

    Zhang, Z.Q.; Fournier, A.; Tan, Y.H.

    1986-06-15

    Radioiodinated human beta-interferon-Ser 17 (Betaseron) was reversibly cross-linked to Daudi cells by dithiobis(succinimidylpropionate). The radioactive ligand was cross-linked to three macromolecules forming labeled complexes of apparent Mr values of 130,000, 220,000, and 320,000. Betaseron, human alpha-interferon, human interleukin 2 but not recombinant human gamma-interferon competed with the labeled ligand for binding to these putative receptor(s). Human leukocyte-produced gamma-interferon competed weakly with /sup 125/I-Betaseron for binding to Daudi cells. The Betaseron-receptor complex(es) was purified by passage through a wheat germ lectin column followed by chromatography on an anti-interferon immunosorbent column and semipreparative gel electrophoresis. The cross-linked ligand-receptor complex was shown to be highly purified by sodium dodecyl sulfate and acetic acid:urea:Triton X-100 polyacrylamide gel electrophoresis. It can be dissociated into the labeled Betaseron (Mr = 17,000) ligand and a receptor moiety which has an apparent molecular weight of 110,000. The chromatographic behavior of the ligand-receptor complex on wheat germ lectin column suggests that the receptor is a glycoprotein. The described procedure yielded about 1 microgram of Betaseron receptor from 10(10) Daudi cells, estimated to contain a maximum of about 15 micrograms of the receptor.

  8. "Old" metal oxide affinity chromatography as "novel" strategy for specific capture of cis-diol-containing compounds.

    PubMed

    Wang, Shao-Ting; Huang, Wei; Deng, Yi-Fan; Gao, Qiang; Yuan, Bi-Feng; Feng, Yu-Qi

    2014-09-26

    The metal oxide affinity chromatography (MOAC) materials have been extensively used for extraction of phosphate compounds in the past decade. Actually, some of these materials also possess adsorption affinity towards cis-diol-containing compounds, which was seldom explored in separation field so far. Here we present the proof-of-concept study to evaluate the feasibility of expanding MOAC for specific capture of cis-diol biomolecules. Benefitting from the high commercialisation of the metal oxide materials, such MOAC strategy possesses several advantages, like synthesis-free, low cost and high expandability. Firstly, the recognition of adenosine against 2'-deoxyadenosine was performed using zirconium oxide and cerium oxide, two typical commercial MOAC materials. The results showed that efficient adsorption and elution could be achieved easily by pH switching from basic to acidic. The isotherm curves demonstrated the adsorption process fitted well with Freundlich isotherm model and was spontaneous at room temperature (ΔG(0)<0) with an exothermic nature (ΔH(0)<0). Afterwards, the highly efficient and selective enrichment of various model cis-diol biomolecules, including ribonucleosides, glycopeptides and glycoproteins, was achieved using this MOAC strategy. Finally, the endogenous ribonucleosides and modified ribonucleosides were successfully purified from human urine sample, which demonstrated the potential application of MOAC materials in the enrichment of target compounds from complex biological samples. Besides the excellent performance of extraction for cis-diol-containing compounds, equally important is that these materials are commercially available with low cost, which makes the MOAC a promising strategy for the study of cis-diol biomolecules in metabolomics and proteomics. PMID:25138708

  9. IDENTIFICATION AND ANALYSIS OF STEREOSELECTIVE DRUG INTERACTIONS WITH LOW DENSITY LIPOPROTEIN BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Sobansky, Matthew R.; Hage, David S.

    2012-01-01

    Columns containing immobilized low density lipoprotein (LDL) were prepared for the analysis of drug interactions with this agent by high-performance affinity chromatography (HPAC). R/S-Propranolol was used as a model drug for this study. The LDL columns gave reproducible binding to propranolol over 60 h of continuous use in the presence of pH 7.4, 0.067 M potassium phosphate buffer. Experiments conducted with this type of column through frontal analysis indicated that two types of interactions were occurring between R-propranolol and LDL, while only a single type of interaction was observed between S-propranolol and LDL. The first type of interaction, which was seen for both enantiomers, involved non-saturable binding; this interaction had an overall affinity (nKa) of 1.9 (± 0.1) × 105 M-1 for R-propranolol and 2.7 (± 0.2) × 105 M-1 for S-propranolol at 37 °C. The second type of interaction was observed only for R-propranolol and involved saturable binding that had an association equilibrium constant (Ka) of 5.2 (± 2.3) × 105 M-1 at 37 °C. Similar differences in binding behavior were found for the two enantiomers at 20 °C and 27 °C. This is the first known example of stereoselective binding of drugs by LDL or other lipoproteins. This work also illustrates the ability of HPAC to be used as a tool for characterizing mixed-mode interactions that involve LDL and related binding agents. PMID:22354572

  10. Characterization of a multiple endogenously expressed Adenosine triphosphate-Binding Cassette transporters using nuclear and cellular membrane affinity chromatography columns

    PubMed Central

    Khadeer, M.A.; Shimmo, R.; Wainer, I.W.; Moaddel, R.

    2014-01-01

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN229)) and (CMAC(LN229)), respectively. Pgp, MRP1and BCRP transporters co-immobilized on both columns was characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs 3.7μM), verapamil (0.6 vs 0.7μM) and prazosin (0.099 vs 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of 8 compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN229) column and decreased it (−5%) on the NMAC(LN229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences. PMID:24642394

  11. TiO2-ZrO2 affinity chromatography polymeric microchip for phosphopeptide enrichment and separation.

    PubMed

    Tsougeni, Katerina; Zerefos, Panagiotis; Tserepi, Angeliki; Vlahou, Antonia; Garbis, Spiros D; Gogolides, Evangelos

    2011-09-21

    We fabricated a TiO(2)-ZrO(2) affinity chromatography micro-column on 2 mm PMMA plates, and demonstrated the enrichment and separation of (a) a standard mono- and tetra-phosphopeptide, and (b) phosphopeptides contained in a tryptic digest of β-Casein. The chromatography column consisted of 32 parallel microchannels with common input and output ports and was fabricated by lithography directly on the polymeric substrate followed by plasma etching (i.e. standard MEMS processing) and sealed with lamination. The liquid deposited TiO(2)-ZrO(2) stationary phase was characterized by X-ray diffraction and was found to be mostly TiO(2) and ZrO(2) in crystalline phases. Off-chip UV detection and MALDI MS identification of the separated effluents were used. The chip had a capacity of >1.4 μg (0.7 nmol) of a prototype mono-phosphopeptide and a recovery of 94 ± 3%, and can be used with small samples (less than 0.1 μL depending on the syringe pump used). The chip design allows an expansion of its capacity by means of increasing the number of parallel microchannels at a constant sample volume. Our approach provided an alternative to off-line extraction tips (with typical capacities of 1-2 μg and sample volumes of 1-10 μL), and to on-chip efforts based on packed bed and frit formats. PMID:21796280

  12. Human plasma alpha-cysteine proteinase inhibitor. Purification by affinity chromatography, characterization and isolation of an active fragment.

    PubMed Central

    Gounaris, A D; Brown, M A; Barrett, A J

    1984-01-01

    Human plasma alpha-cysteine proteinase inhibitor (alpha CPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high-resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64 000 and material of higher Mr (about 100 000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of Mr 64 000. An antiserum was raised against alpha CPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9 mg/dl. Both low-Mr and high-Mr forms of alpha CPI were confirmed to be sialoglycoproteins by the decrease in electrophoretic mobility after treatment with neuraminidase. alpha CPI was shown immunologically to be distinct from antithrombin III and alpha 1-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. alpha CPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of alpha CPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. alpha CPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34 pM, 1.1 nM and 62 pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 microM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of alpha CPI with bromelain gave rise to an inhibitory fragment of Mr about 22 000, which was isolated. Images Fig. 2. Fig. 3. Fig. 4. PMID:6548132

  13. Studies on human pregnancy-associated plasma protein A. Purification by affinity chromatography and structural comparisons with alpha 2-macroglobulin.

    PubMed Central

    Sutcliffe, R G; Kukulska-Langlands, B M; Coggins, J R; Hunter, J B; Gore, C H

    1980-01-01

    Pregnancy-associated plasma protein-A (PAPP-A) has been purified by a combination of methods including antibody-affinity chromatography. The resultant protein, obtained in 16% yield from maternal serum, appeared as a single major component on non-denaturing polyacrylamide and SDS/polyacrylamide gel electrophoresis. The protein showed a single component when analysed by isoelectric focusing under denaturing conditions in the presence and absence of reduction and had a pI of 4.34 and 4.42 respectively. These pI values were indistinguishable from those of alpha 2-macroglobulin (alpha 2M). The molecular weight of the PAPP-A polypeptide as shown by SDS/polyacrylamide-gel electrophoresis was 187000, with a minor component of mol.wt. 82500 that was attributed to proteolysis. Since native PAPP-A had a molecular weight on gel chromatography very similar to that of alpha 2M (620000--820000), it was concluded that PAPP-A was a homotetramer. In the absence of reduction, a high-molecular-weight (420000) protomer of PAPP-A was found. It was deduced that PAPP-A, like alpha 2M, is a dinner, whose protomers are composed of disulphide-linked polypeptide chains. It was found that the molecular weight of the PAPP-A polypeptide exceeded that of alpha 2M by 3.3%, but that the total carbohydrate content of PAPP-A exceeded that of alpha 2M by 10% and that its neutral carbohydrate content exceeded that of alpha 2M by between 7.4 and 9.0%. The significance of the estimated molecular weights of alpha 2M (181000) and its major tryptic fragments is discussed in the light of published values. A tryptic fragment alpha 2M (82500 mol.wt.) was apparently the same size as the major tryptic fragment of PAPP-A. Images Fig. 1. Fig. 4. Fig. 6. PMID:6169340

  14. Further characterization of the low and high affinity binding components of the thyrotropin receptor

    SciTech Connect

    McQuade, R.; Thomas, C.G. Jr.; Nayfeh, S.N.

    1986-05-29

    Following cross-linking with disuccinimdiyl suberate and analysis by SDS-PAGE and autoradiography, both the high- and low-affinity TSH binding components exhibited two similar /sup 125/I-TSH-labeled bands, with Mr values of 80,000 and 68,000. IgG fractions from patients with Graves' disease inhibited /sup 125/I-TSH binding to both components, while normal IgG had no effect. Although not entirely conclusive, these results suggest that the high- and low-affinity components share similar subunit composition and antigenic determinants.

  15. Affinity crosslinking of /sup 125/I-human beta-endorphin to cell lines possessing either mu or delta type opioid binding sites

    SciTech Connect

    Keren, O.; Gioannini, T.L.; Hiller, J.M.; Simon, E.J.

    1986-01-01

    Affinity crosslinking of human /sup 125/I-beta-Endorphin to cell lines possessing either mu or delta binding sites was carried out. Autoradiography of SDS-PAGE gels from these crosslinked cell lines revealed that these two sites contain major peptide subunits that differ in molecular size. This confirms our earlier finding in mammalian brain which demonstrated separate and distinct subunits for mu and delta opioid receptors.

  16. Characterization of a Multiple Ligand-Gated Ion Channel Cellular Membrane Affinity Chromatography Column and Identification of Endogenously Expressed Receptors in Astrocytoma Cell Lines

    PubMed Central

    Kitabatake, T.; Moaddel, R.; Cole, R.; Gandhari, M.; Frazier, C.; Hartenstein, J.; Rosenberg, A.; Bernier, M.; Wainer, I. W.

    2008-01-01

    Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [3H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric α7 nicotinic acetylcholine receptors (α7 nAChR) and heteromeric nicotinic acetylcholine receptors (αxβy nAChRs), which was confirmed by the addition of subtype-specific inhibitors, κ-bungarotoxin (α7 nAChR) and K-bungarotoxin (αxβy nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), γ-aminobutyric acid (GABAA) and N-methyl-d-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABAA receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors. PMID:18847217

  17. Characterization of a multiple ligand-gated ion channel cellular membrane affinity chromatography column and identification of endogenously expressed receptors in astrocytoma cell lines.

    PubMed

    Kitabatake, T; Moaddel, R; Cole, R; Gandhari, M; Frazier, C; Hartenstein, J; Rosenberg, A; Bernier, M; Wainer, I W

    2008-11-15

    Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [(3)H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric alpha7 nicotinic acetylcholine receptors (alpha7 nAChR) and heteromeric nicotinic acetylcholine receptors (alpha(x)beta(y) nAChRs), which was confirmed by the addition of subtype-specific inhibitors, alpha-bungarotoxin (alpha7 nAChR) and kappa-bungarotoxin (alpha(x)beta(y) nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), gamma-aminobutyric acid (GABA(A)) and N-methyl-D-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABA(A) receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors. PMID:18847217

  18. Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

    PubMed

    Chen, Chen; Khoury, Graziella El; Lowe, Christopher R

    2014-10-15

    One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7mg GOx/ml resin and an apparent dissociation constant (Kd) of 1.45×10(-6)M at pH 7.4. The adsorbent can also bind 8.1mg AGP/ml resin and displays an apparent affinity constant Kd=1.44×10(-5)M. The ligand has a sugar specificity in the following sequence: sorbitol>fructose>mannitol>ribose>arabinose>xylose>galactose>mannose>glucose>fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and

  19. Liver- and bone-derived isoenzymes of alkaline phosphatase in serum as determined by high-performance affinity chromatography.

    PubMed

    Anderson, D J; Branum, E L; O'Brien, J F

    1990-02-01

    To separate liver and bone alkaline phosphatase (ALP) isoenzymes in human serum, we used high-performance affinity chromatography (HPAC) on a column of wheat-germ lectin conjugated to 7-microns-diameter silica particles and an eluent containing N-acetyl-D-glucosamine (NAG). On-line spectrophotometric detection of ALP involved pumping diethanolamine-buffered p-nitrophenyl phosphate solution post-column. Bone and liver isoenzymes could be separated into two peaks with only 10% overlap when an exponential gradient was used. A linear-step gradient separated 80.9% of liver ALP and 91.6% of bone ALP in two distinct peaks. True bone and liver ALP peak areas for the linear-step gradient were determined by using correction factors, because each peak contained a co-eluted portion of the other ALP isoenzyme. The detection limit improved 10-fold over those of other techniques for ALP isoenzymes, owing to the relatively large sample that could be applied to the column. Correlation with a urea-inactivation procedure was reasonable for patients' serum samples (r = 0.98 and 0.79 for liver ALP and bone ALP, respectively). PMID:2302767

  20. Biotin-functionalized poly(ethylene terephthalate) capillary-channeled polymer fibers as HPLC stationary phase for affinity chromatography.

    PubMed

    Jiang, Liuwei; Marcus, R Kenneth

    2015-01-01

    Native poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers have been used as the stationary phase for high-performance liquid chromatography (HPLC) of proteins via reversed-phase and ion-exchange processes. Functionalization can be used to bring about greater selectivity through surface modification. PET fibers were treated with ethylenediamine to generate primary amine groups on the fiber surface, enabling subsequent covalent attachment of ligands. The ninhydrin test for primary amines revealed surface densities of 13.9-60.0 μmol m(-2) for PET fibers exposed for periods of 3-12 min. Here, 8-amino-3,6-dioxaoctanoic acid was linked to the EDA-treated PET fiber surface as a hydrophilic spacer, and then D-biotin was attached on the end of the spacer as an affinity ligand. The streptavidin binding capacity and binding homogeneity were studied on the biotin-functionalized PET C-CP fiber microbore column. The selectivity of the biotin surface functionalization was assessed by spiking lysate with Texas Red-labeled streptavidin and enhanced green fluorescent protein. Greater than 99% selectivity was realized. This ligand-coupling strategy from standard solid-phase peptide synthesis used in stationary phase functionalization creates great potential for PET C-CP fiber-packed HPLC columns to perform a variety of chromatographic separations. PMID:25410640

  1. Glycopeptide Site Heterogeneity and Structural Diversity Determined by Combined Lectin Affinity Chromatography/IMS/CID/MS Techniques

    NASA Astrophysics Data System (ADS)

    Zhu, Feifei; Trinidad, Jonathan C.; Clemmer, David E.

    2015-07-01

    Glycopeptides from a tryptic digest of chicken ovomucoid were enriched using a simplified lectin affinity chromatography (LAC) platform, and characterized by high-resolution mass spectrometry (MS) as well as ion mobility spectrometry (IMS)-MS. The LAC platform effectively enriched the glycoproteome, from which a total of 117 glycopeptides containing 27 glycan forms were identified for this protein. IMS-MS analysis revealed a high degree of glycopeptide site heterogeneity. Comparison of the IMS distributions of the glycopeptides from different charge states reveals that higher charge states allow more structures to be resolved. Presumably the repulsive interactions between charged sites lead to more open configurations, which are more readily separated compared with the more compact, lower charge state forms of the same groups of species. Combining IMS with collision induced dissociation (CID) made it possible to determine the presence of isomeric glycans and to reconstruct their IMS profiles. This study illustrates a workflow involving hybrid techniques for determining glycopeptide site heterogeneity and evaluating structural diversity of glycans and glycopeptides.

  2. Purification of His6-organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography.

    PubMed

    Efremenko, E; Votchitseva, Y; Plieva, F; Galaev, I; Mattiasson, B

    2006-05-01

    Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His6-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me2+-iminodiacetic acid (IDA)-polyacrylamide cryogel (PAA) and Me2+-N,N,N'-tris (carboxymethyl) ethylendiamine (TED)-PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA-PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co2+-IDA-PAA provided the highest level of selectivity for His6-OPH. Comparative analysis of purification using Co2+-IDA-PAA and Ni-nitrilotriacetic acid-agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration. PMID:16088350

  3. Process characterization for metal-affinity chromatography of an Fc fusion protein: a design-of-experiments approach.

    PubMed

    Shukla, A A; Sorge, L; Boldman, J; Waugh, S

    2001-10-01

    The utility of a design-of-experiments approach was investigated for process characterization of a metal-affinity chromatographic purification process for an Fc fusion protein. This approach gave a better understanding of some of the key process variables as well as robustness for this step in the purification process. Single-variable experiments were employed to screen some of the potentially important variables in this step. Ranges for these variables were set based on prior experience in clinical manufacturing with similar processes. Following these experiments, a fractional factorial study was employed to further investigate the most important variables and their interactions. Key operational variables that had an impact on step yield and eluate purity were identified. In addition, the study helped identify a worst-case scenario for the step purity and helped assure that the rest of the process would successfully purify the product. This paper demonstrates the utility of a design-of-experiments approach for the characterization and validation of process chromatography steps in downstream processing. In addition, this study emphasizes the utility of robustness studies early in process development and establishes a strategy for future robustness studies. PMID:11592911

  4. The identification by affinity chromatography of the rat liver ribosomal proteins that bind to elongator and initiator transfer ribonucleic acids.

    PubMed

    Ulbrich, N; Wool, I G; Ackerman, E; Sigler, P B

    1980-07-25

    Mixed yeast elongator-tRNAs (bulk tRNA lacking fRNAm,fMet), pure isoaccepting species of elongator-tRNAs (tRNAmMet and tRNAPhe), and purified initiator-tRNA (tRNAfMet) were each oxidized with periodate and the 3' terminus was coupled to Sepharose 4B through an adipic acid dihydrazide spacer. The rat liver ribosomal proteins that associated with the tRNAs were isolated by affinity chromatography and identified by electrophoresis in polyacrylamide gels. The rat liver ribosomal proteins that were bound to the elongator-tRNA preparations were L6, L35a, and S15; small amounts of a number of other proteins also associated with the nucleic acid. When initiator-tRNA (tRNAfMet) was immobilized on Sepharose, only L6 and L35a were bound; no 40 S subunit proteins associated with initiator-tRNA. No Escherichia coli proteins formed a complex with either eukaryotic initiator- or elongator-tRNAs. PMID:7391064

  5. Inosine 5'-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5'-monophosphate.

    PubMed Central

    Gilbert, H J; Lowe, C R; Drabble, W T

    1979-01-01

    Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli. PMID:44191

  6. Wide Range of Biotin (Vitamin H) Content in Foodstuffs and Powdered Milks as Assessed by High-performance Affinity Chromatography

    PubMed Central

    Hayakawa, Kou; Katsumata, Noriyuki; Abe, Kiyomi; Hirano, Masahiko; Yoshikawa, Kazuyuki; Ogata, Tsutomu; Horikawa, Reiko; Nagamine, Takeaki

    2009-01-01

    The biotin (vitamin H) contents of various foodstuffs were determined by using a newly developed high-performance affinity chromatography with a trypsin-treated avidin-bound column. Biotin was derivatized with 9-anthryldiazomethane (ADAM) to fluorescent biotin-ADAM ester. A wide range of biotin contents were found in various foodstuffs depending upon the species (strain), season, organ (of plants and animals), geography, freshness, preparation method and storage method. Among the foodstuffs and fermented foods tested, it was found that wide distributions of biotin content were observed in powdered milk, natto, sake (rice wine), beer, edible oil and sea weed. Since powdered milk is important for child health and development, 14 kinds of powdered and special milks for use in children’s diseases were intensively measured. We found that several special milk powders for children with allergies contained low levels of free biotin. Use of these powdered milks caused skin diseases and alopecia in some patients possessing thermolabile serum biotinidase, and administration of free biotin improved their symptoms dramatically. Therefore, it is essential to estimate the total and free biotin contents on each foodstuff in order to improve effective biotin intake and support better health and quality of life for people. PMID:24790379

  7. Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum.

    PubMed

    Smits, Nathalie G E; Blokland, Marco H; Wubs, Klaas L; Nessen, Merel A; van Ginkel, Leen A; Nielen, Michel W F

    2015-08-01

    The use of recombinant bovine somatotropin (rbST) to enhance milk production is approved in several countries, but it is prohibited in the European Union. According to EU legislation, it is necessary to confirm positive screening results prior to enforcement. Although adequate screening assays are available nowadays, development of liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory methods to detect low levels of rbST is still a challenge. Here, we present a novel approach using immuno-affinity enrichment on monolithic micro-columns in combination with state-of-the-art ultra-high pressure LC-MS/MS (UHPLC-MS/MS) detection. The developed approach enables detection and confirmation of rbST in serum at a decision limit (CCα) concentration of 0.8 ng mL(-1). Furthermore, the method is easy to handle, robust and reproducible. We successfully applied the confirmatory method to serum samples from rbST treated cows that were found suspect after immunoassay-based screening. The use of rbST could be confirmed over 1 week after treatment, and the developed method demonstrated the sensitivity needed for effective control. Graphical Abstract Graphical summary of the workflow, for serum preparation, enrichment with monolith microcolumns and LC-MS/MS measurement of rbST. PMID:26077745

  8. Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry

    SciTech Connect

    Niture, Suryakant K.; Doneanu, Catalin E.; Velu, Chinavenmeni S.; Bailey, Nathan I.; Srivenugopal, Kalkunte S. . E-mail: Kalkunte.srivenugopal@ttuhsc.edu

    2005-12-02

    Recent evidence suggests that human O {sup 6}-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT-Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase {delta}, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21{sup waf1/cip1}), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1{alpha}), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90{alpha} and {beta}, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.

  9. Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography

    PubMed Central

    Machado, Gleyce Alves; de Oliveira, Heliana Batista; Gennari-Cardoso, Margareth Leitão; Mineo, José Roberto; Costa-Cruz, Julia Maria

    2013-01-01

    The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (Junbound) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJunbound) and aqueous (AJunbound) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for Junbound, 92.5% and 93.5% for DJunboundand 82.5% and 82.6% for AJunbound. By immunoblot, the DJunboundfraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJunboundfraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot. PMID:23778661

  10. [Affinity chromatography and proteomic screening as the effective method for S100A4 new protein targets discovery].

    PubMed

    Koshelev, Iu A

    2014-01-01

    Affinity chromatography followed by a selective binding proteins identification can be using as effective method for a biological impotent interactions discovery. The molecular structure and their surface charge as and conformational regulation possibilities, which change their surface hydrophobic properties, all they should to taken in account during method optimization process. With the same' method we had identify some new S100A4 target proteins such as cytoskeleton proteins Sept2, Sept7, Sept11 and this interaction would can to highlight as S100A4 would regulate cell motility. Even we had identify the transcription cofactor Ddx5 and through such complex formation a S100A4 protein would can to regulate E-cadherin, p21 Waf1/Cip1), Bnip3 gene expression. The same protocol can be using for a target proteins search with another S100 protein family members, because their molecules demonstrate a high homology level in amino aside sequences and 3D structures. PMID:25842873

  11. LC–MS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography

    PubMed Central

    2015-01-01

    Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC–MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC–ESI–MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts. PMID:25134008

  12. Glycopeptide Site Heterogeneity and Structural Diversity Determined by Combined Lectin Affinity Chromatography/IMS/CID/MS Techniques.

    PubMed

    Zhu, Feifei; Trinidad, Jonathan C; Clemmer, David E

    2015-07-01

    Glycopeptides from a tryptic digest of chicken ovomucoid were enriched using a simplified lectin affinity chromatography (LAC) platform, and characterized by high-resolution mass spectrometry (MS) as well as ion mobility spectrometry (IMS)-MS. The LAC platform effectively enriched the glycoproteome, from which a total of 117 glycopeptides containing 27 glycan forms were identified for this protein. IMS-MS analysis revealed a high degree of glycopeptide site heterogeneity. Comparison of the IMS distributions of the glycopeptides from different charge states reveals that higher charge states allow more structures to be resolved. Presumably the repulsive interactions between charged sites lead to more open configurations, which are more readily separated compared with the more compact, lower charge state forms of the same groups of species. Combining IMS with collision induced dissociation (CID) made it possible to determine the presence of isomeric glycans and to reconstruct their IMS profiles. This study illustrates a workflow involving hybrid techniques for determining glycopeptide site heterogeneity and evaluating structural diversity of glycans and glycopeptides. PMID:25840811

  13. ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: BINDING OF GLIBENCLAMIDE TO NORMAL AND GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2012-01-01

    High-performance affinity chromatography (HPAC) was used to examine the changes in binding that occur for the sulfonylurea drug glibenclamide with human serum albumin (HSA) at various stages of glycation for HSA. Frontal analysis on columns containing normal HSA or glycated HSA indicated glibenclamide was interacting through both high affinity sites (association equilibrium constant, Ka, 1.4–1.9 × 106 M−1 at pH 7.4 and 37°C) and lower affinity sites (Ka, 4.4–7.2 × 104 M−1). Competition studies were used to examine the effect of glycation at specific binding sites of HSA. An increase in affinity of 1.7- to 1.9-fold was seen at Sudlow site I with moderate to high levels of glycation. An even larger increase of 4.3- to 6.0-fold in affinity was noted at Sudlow site II for all of the tested samples of glycated HSA. A slight decrease in affinity may have occurred at the digitoxin site, but this change was not significant for any individual glycated HSA sample. These results illustrate how HPAC can be used as tool for examining the interactions of relatively non-polar drugs like glibenclamide with modified proteins and should lead to a more complete understanding of how glycation can alter the binding of drugs in blood. PMID:23092871

  14. Purification of human immunoglobulins A, G and M from Cohn fraction II/III by small peptide affinity chromatography.

    PubMed

    Liu, Zhuo; Gurgel, Patrick V; Carbonell, Ruben G

    2012-11-01

    This work describes attempts to purify human IgG, IgA and IgM from Cohn fraction II/III using HWRGWV affinity peptide resin. The effects of peptide density and different elution additives on recovery of the three antibodies were investigated. At low peptide density, salting-in salts such as magnesium chloride and calcium chloride facilitated antibody elution. Ethylene glycol, urea and arginine also facilitated elution because of their ability to decrease hydrophobic interactions, hydrogen bonding and electrostatic interactions. However, at high peptide density, no recovery improvements were observed because of increased non-specific hydrophobic interactions. The final elution conditions for each antibody were chosen based on the resulting yields and purities when a 10:2:1mg/mL mixture of human IgG, IgA and IgM was used as starting material. Different pretreatment methods were employed in order to improve the purity of antibodies from Cohn fraction II/III. After pretreatment with caprylic acid precipitation or combination of caprylic acid and polyethylene glycol precipitation, purities over 95% and yields of about 60% were obtained for hIgG, which are comparable to current chromatographic purification methods involving two chromatography steps when hIgG is isolated from plasma fractions. A hIgA-enriched fraction with 42% hIgA and 56% hIgG, as well as a hIgM enriched fraction with 46% hIgM, 28% hIgA and 24% hIgG, were obtained as the by-products. PMID:23026261

  15. Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

    PubMed Central

    Kaur, Jasvir; Reinhardt, Dieter P.

    2012-01-01

    Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. PMID:23119075

  16. Affinity chromatography of alpha/sub 2/-adrenergic receptors (. cap alpha. /sub 2/AR) from pig cerebral cortex

    SciTech Connect

    Repaske, M.G.; Limbird, L.E.

    1986-03-01

    A high capacity, ..cap alpha../sub 2/AR-selective affinity resin (YOH. ag) has been prepared by coupling yohimbinic acid to diaminodipropylamine agarose with 1,3 dicyclohexylcarbodiimide. Unreacted amino groups on the agarose matrix are blocked subsequently by acetylation. One volume of YOH. ag adsorbs 75% of the ..cap alpha../sub 2/AR from 50 volumes of digitonin-solubilized preparation containing 0.2 pmol ..cap alpha../sub 2/AR/mg protein. Digitonin-solubilized preparations are derived from cholate extracts of porcine cerebral cortex containing approx. 0.075 pmol ..cap alpha../sub 2/AR/mg protein. Adsorption of ..cap alpha../sub 2/AR to YOH. ag is selective and thus is blocked by the ..cap alpha..-adrenergic antagonist phentolamine. Adsorbed ..cap alpha../sub 2/AR are eluted with 10 ..mu..M phentolamine (20% yield) after removal of non-related proteins with NaCl gradients. Following hydroxylapatite chromatography to concentrate ..cap alpha..''AR and to remove phentolamine, the ..cap alpha..AR is present at 200-400 pmol/mg protein, assayed using sub-saturating concentrations of (/sup 3/H)-yohimbine. (It is estimated that the specific activity of a homogeneous ..cap alpha../sub 2/AR preparation would be 12,000-16,000 pmol/mg protein.) The availability of large quantities of cortical ..cap alpha../sub 2/AR and a resin easily prepared from commercially-supplied reagents suggests that purification of quantities of ..cap alpha../sub 2/AR sufficient for subsequent biochemical studies is feasible.

  17. Using Affinity Chromatography to Investigate Novel Protein–Protein Interactions in an Undergraduate Cell and Molecular Biology Lab Course

    PubMed Central

    2009-01-01

    Inquiry-driven lab exercises require students to think carefully about a question, carry out an investigation of that question, and critically analyze the results of their investigation. Here, we describe the implementation and assessment of an inquiry-based laboratory exercise in which students obtain and analyze novel data that contribute to our understanding of macromolecular trafficking between the nucleus and cytoplasm in eukaryotic cells. Although many of the proteins involved in nucleocytoplasmic transport are known, the physical interactions between some of these polypeptides remain uncharacterized. In this cell and molecular biology lab exercise, students investigate novel protein–protein interactions between factors involved in nuclear RNA export. Using recombinant protein expression, protein extraction, affinity chromatography, SDS-polyacrylamide gel electrophoresis, and Western blotting, undergraduates in a sophomore-level lab course identified a previously unreported association between the soluble mRNA transport factor Mex67 and the C-terminal region of the yeast nuclear pore complex protein Nup1. This exercise immersed students in the process of investigative science, from proposing and performing experiments through analyzing data and reporting outcomes. On completion of this investigative lab sequence, students reported enhanced understanding of the scientific process, increased proficiency with cellular and molecular methods and content, greater understanding of data analysis and the importance of appropriate controls, an enhanced ability to communicate science effectively, and an increased enthusiasm for scientific research and for the lab component of the course. The modular nature of this exercise and its focus on asking novel questions about protein–protein interactions make it easily transferable to undergraduate lab courses performed in a wide variety of contexts. PMID:19723816

  18. Engineering foot-and-mouth disease virus serotype O IND R2/1975 for one-step purification by immobilized metal affinity chromatography.

    PubMed

    Biswal, Jitendra K; Bisht, Punam; Subramaniam, Saravanan; Ranjan, Rajeev; Sharma, Gaurav K; Pattnaik, Bramhadev

    2015-09-01

    Immobilized metal affinity chromatography (IMAC) allows for the efficient protein purification via metal affinity tag such as hexa-histidine (His6) sequence. To develop a new chromatography strategy for the purification and concentration of foot-and-mouth disease virus (FMDV) particles, we inserted the His6-tag at the earlier reported site in the VP1 G-H loop of the FMD virus serotype O vaccine strain IND R2/1975. Display of the His6-tag on the capsid surface, endowed the virus with an increased affinity for immobilized nickel ions. We demonstrated that the His6-tagged FMDV could be produced to high titre and purified from the infected BHK-21 cell lysates by IMAC efficiently. Further, a 1150-fold reduction in protein contaminant level and an 8400-fold reduction in DNA contaminant level were achieved in the IMAC purification of His6-tagged FMDV. Through various functional assays it has been found that the tagged virus retains its functionality and infectivity similar to the non-tagged virus. The affinity purification of the His6-tagged FMDV may offer a feasible, alternative approach to the current methods of FMDV antigen purification, concentration and process scalability. PMID:26123433

  19. Chromatography

    MedlinePlus

    Chromatography is a way of separating two or more chemical compounds. Chemical compounds are chemicals that are ... of chemical compound. There are different kinds of chromatography. These include gas, high pressure liquid, or ion ...

  20. Isolation and purification of recombinant human plasminogen Kringle 5 by liquid chromatography and ammonium sulfate salting-out.

    PubMed

    Bian, Liujiao; Ji, Xu; Hu, Wei

    2014-07-01

    In this work, a novel method was established to isolate and purify Human plasminogen Kringle 5 (HPK5) as a histidine-tagged fusion protein expressed in Escherichia coli BL21 (DE3). This method consisted of sample extraction using a Ni-chelated Sepharose Fast-Flow affinity column, ammonium sulfate salting-out and Sephadex G-75 size-exclusion column in turn. The purity analysis by SDS-PAGE, high-performance size-exclusion and reversed-phase chromatographies showed that the obtained recombinant fusion HPK5 was homogeneous and its purity was higher than 96%; the activity analysis by chorioallantoic membrane model of chicken embryos revealed that the purified recombinant HPK5 exhibited an obvious anti-angiogenic activity under the effective range of 5.0-25.0 µg/mL. Through this procedure, about 19 mg purified recombinant fusion HPK5 can be obtained from 1 L of original fermentation solution. Approximate 32% of the total recombinant fusion HPK5 can be captured and the total yield was approximately 11%. PMID:24311387

  1. Screening and confirmation of thyreostatics in urine by gas chromatography with nitrogen-phosphorus detection and gas chromatography-mass spectrometry after sample clean-up with a mercurated affinity column.

    PubMed

    Schilt, R; Weseman, J M; Hooijerink, H; Korbee, H J; Traag, W A; van Steenbergen, M J; Haasnoot, W

    1989-04-01

    Methods are described for the screening and confirmation of residues of the thyreostatics thiouracil, methylthiouracil and propylthiouracil in urine samples of cattle at levels down to 25 micrograms/l. After a selective preconcentration of the thiol-containing thyreostatics on a mercurated affinity column, the analytes are derivatized by extractive alkylation and analysed by gas chromatography with nitrogen-phosphorus or mass spectrometric detection. PMID:2745644

  2. Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr-activated sepharose-4B affinity chromatography.

    PubMed

    Hedayati Ch, Mojtaba; Amani, Jafar; Sedighian, Hamid; Amin, Mohsen; Salimian, Jafar; Halabian, Raheleh; Imani Fooladi, Abbas Ali

    2016-09-01

    Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA-aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (kd  = 2.3 × 10(-11) ). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27091327

  3. Simplifying the synthesis of SIgA: combination of dIgA and rhSC using affinity chromatography

    PubMed Central

    Moldt, Brian; Saye-Francisco, Karen; Schultz, Niccole; Burton, Dennis R.; Hessell, Ann J.

    2013-01-01

    The mucosal epithelia together with adaptive immune responses, such as local production and secretion of dimeric and polymeric immunoglobulin A (IgA), are a crucial part of the first line of defense against invading pathogens. IgA is primarily secreted as SIgA and plays multiply roles in mucosal defense. The study of SIgA-mediated protection is an important area of research in mucosal immunity but an easy, fast and reproducible method to generate pathogen-specific SIgA in vitro has not been available. We report here a new method to produce SIgA by co-purification of dimeric IgA, containing J chain, and recombinant human SC expressed in CHO cells. We previously reported the generation, production and characterization of the human recombinant monoclonal antibody IgA2 b12. This antibody, derived from the variable regions of the neutralizing anti-HIV-1 mAb IgG1 b12, blocked viral attachment and uptake by epithelial cells in vitro. We used a cloned CHO cell line that expresses monomeric, dimeric and polymeric species of IgA2 b12 for large-scale production of dIgA2 b12. Subsequently, we generated a CHO cell line to express recombinant human secretory component (rhSC). Here, we combined dIgA2 b12 and CHO-expressed rhSC via column chromatography to produce SIgA2 b12 that remains fully intact upon elution with 0.1M Citric acid, pH 3.0. We have performed biochemical analysis of the synthesized SIgA to confirm the species is of the expected size and retains the functional properties previously described for IgA2 b12. We show that SIgA2 b12 binds to the HIV-1 gp120 glycoprotein with similar apparent affinity to that of monomeric and dimeric forms of IgA2 b12 and neutralizes HIV-1 isolates with similar potency. An average yield of 6 mg of SIgA2 b12 was achieved from the combination of 20 mg of purified dIgA2 b12 and 2 L of rhSC-containing CHO cell supernatant. We conclude that synthesized production of stable SIgA can be generated by co-purification. This process introduces a

  4. Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns.

    PubMed

    Bi, Cong; Zheng, Xiwei; Hage, David S

    2016-02-01

    In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110-830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10-20min when using only 3-10μL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum. PMID:26797422

  5. Analysis of multi-site drug-protein interactions by high-performance affinity chromatography: Binding by glimepiride to normal or glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S

    2015-08-21

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2-11.8×10(5)M(-1) at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9-16×10(3)M(-1)). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  6. Analysis of Multi-Site Drug-Protein Interactions by High-Performance Affinity Chromatography: Binding by Glimepiride to Normal or Glycated Human Serum Albumin

    PubMed Central

    Matsuda, Ryan; Li, Zhao; Zheng, Xiwei; Hage, David S.

    2015-01-01

    High-performance affinity chromatography (HPAC) was used in a variety of formats to examine multi-site interactions between glimepiride, a third-generation sulfonylurea drug, and normal or in vitro glycated forms of the transport protein human serum albumin (HSA). Frontal analysis revealed that glimepiride interacts with normal HSA and glycated HSA at a group of high affinity sites (association equilibrium constant, or Ka, 9.2–11.8 × 105 M−1 at pH 7.4 and 37°C) and a group of lower affinity regions (Ka, 5.9–16.2 × 103 M−1). Zonal elution competition studies were designed and carried out in both normal- and reversed-role formats to investigate the binding by this drug at specific sites. These experiments indicated that glimepiride was interacting at both Sudlow sites I and II. Allosteric effects were also noted with R-warfarin at Sudlow site I and with tamoxifen at the tamoxifen site on HSA. The binding at Sudlow site I had a 2.1- to 2.3-fold increase in affinity in going from normal HSA to the glycated samples of HSA. There was no significant change in the affinity for glimepiride at Sudlow site II in going from normal HSA to a moderately glycated sample of HSA, but a slight decrease in affinity was seen in going to a more highly glycated HSA sample. These results demonstrated how various HPAC-based methods can be used to profile and characterize multi-site binding by a drug such as glimepiride to a protein and its modified forms. The information obtained from this study should be useful in providing a better understanding of how drug-protein binding may be affected by glycation and of how separation and analysis methods based on HPAC can be employed to study systems with complex interactions or that involve modified proteins. PMID:26189669

  7. HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND THE ANALYSIS OF DRUG INTERACTIONS WITH MODIFIED PROTEINS: BINDING OF GLICLAZIDE WITH GLYCATED HUMAN SERUM ALBUMIN

    PubMed Central

    Matsuda, Ryan; Anguizola, Jeanethe; Joseph, K.S.; Hage, David S.

    2011-01-01

    This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (Ka) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high affinity sites (average Ka, 7.1-10 × 104 M−1) and a group of lower affinity sites (average Ka, 5.7-8.9 × 103 M−1) at pH 7.4 and 37°C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the Ka values for gliclazide at these sites being 1.9 × 104 M−1 and 6.0 × 104 M−1, respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification. PMID:21922305

  8. Affinity chromatography using 2' fluoro-substituted RNAs for detection of RNA-protein interactions in RNase-rich or RNase-treated extracts.

    PubMed

    Hovhannisyan, Ruben; Carstens, Russ

    2009-02-01

    Use of RNA affinity chromatography is commonly used to identify RNA binding proteins that interact with specific RNA cis-elements that function in post-transcriptional gene regulation. These purifications can be complicated by residual RNase activity in cellular extracts that can degrade the RNAs on these affinity columns. Furthermore, some proteins may associate indirectly with the column as a component of multi-protein complexes that are "tethered" through the binding of cellular RNAs. We present a protocol for an RNA affinity procedure that can be used in conjunction with RNase-rich or RNase-treated extracts by using RNAs synthesized with 2' fluoro-substituted cytidine triphosphate (CTP) and uridine triphosphate (UTP). The resulting RNAs are shown to be RNase A-resistant and capable of direct coupling to adipic acid dihydrazide agarose beads. Using an RNA cis-element previously shown to bind hnRNP M, we demonstrated that the substituted RNAs preserve binding capability by a common class of RNA binding proteins. Our results provide a method that may be used more generally for RNA affinity purification or as a validation step to verify more direct binding of a given RNA binding protein to a target RNA. PMID:19317654

  9. Phosphatidylglycerol biosynthesis in Bacillus licheniformis Resolution of membrane-bound enzymes by affinity chromatography on cytidinediphospho-sn-1,2-diacylglycerol Sepharose.

    PubMed

    Larson, T J; Hirabayshi, T; Dowhan, W

    1976-03-01

    Cytidinediphospho-sn-1,2-diaclglycerol (CDP-diglyceride) has been covalently linked to Sephrose 4B via adipic acid dihydrazide spacer arm forming an effective affinity chromatography column. This liponucleo-tide ligand and sn-glycero-3-phosphate are subtracts for the formation of 3-sn-phoshatidyl-1'-sn-glycero-3'-phosphate (PGP) catalyzed in both eukaryotic and prokaryotic organisms by sn-glycero-3-phosphate: CMP phosphatidlytranferase (PGP synthetase). Using this CDP-diglyceride Sephrose affinity column we were able to resolve the membrane associated 3-sn-phosphatidyl'1-sn-glycerol (PG) synthesizing system present in Bacillus licheniformis into two activities. A PGP synthetase activity was adsorbed to the affinity column and was eluted using buffer containg CDP-diglyceride; a PGP phosphatease acactivity had no affinity for the column. Both PGP synthase and PGP phosphatase of B. licheniformis were associated with a membrane component of the cell as evidenced by sucrose gradient centrifugation, differential centrifugation, and solubilization by buffers containing detergent... PMID:175832

  10. Engineering Escherichia coli BL21(DE3) Derivative Strains To Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography ▿ † ‡

    PubMed Central

    Robichon, Carine; Luo, Jianying; Causey, Thomas B.; Benner, Jack S.; Samuelson, James C.

    2011-01-01

    Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The “NiCo” strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein. PMID:21602383

  11. Engineering of a metal coordinating site into human glutathione transferase M1-1 based on immobilized metal ion affinity chromatography of homologous rat enzymes.

    PubMed

    Chaga, G; Widersten, M; Andersson, L; Porath, J; Danielson, U H; Mannervik, B

    1994-09-01

    Rat glutathione transferase (GST) 3-3 binds to Ni(II)-iminodiacetic acid (IDA)-agarose, whereas other GSTs that are abundant in rat liver do not bind to this immobilized metal ion affinity chromatography (IMAC) adsorbent. Rat GST 3-3 contains two superficially located amino acid residues, His84 and His85, that are suitably positioned for coordination to Ni(II)-IDA-agarose. This particular structural motif is lacking in GSTs that do not bind to the IMAC matrix. Creation of an equivalent His-His structure in the homologous human GST M1-1 by protein engineering afforded a mutant enzyme that displays affinity for Ni(II)-IDA-agarose, in contrast to the wild-type GST M1-1. The results identify a distinct site that is operational in IMAC and suggest an approach to the rational design of novel integral metal coordination sites in proteins. PMID:7831282

  12. Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

    PubMed

    Marchenko, N Iu; Marchenkov, V V; Kaĭsheva, A L; Kashparov, I A; Kotova, N V; Kaliman, P A; Semisotnov, G V

    2006-12-01

    The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions. PMID:17223789

  13. Use of differential dye-ligand chromatography with affinity elution for enzyme purification: 2-keto-3-deoxy-6-phosphogluconate aldolase from Zymomonas mobilis.

    PubMed

    Scopes, R K

    1984-02-01

    2-Keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) has been isolated from extracts of Zymomonas mobilis using differential dye-ligand chromatography and affinity elution with product/product analog. The one-step procedure gives an enzyme with specific activity 600 units mg-1. Only 1 out of 47 dyes, Procion Yellow MX-GR, bound the enzyme completely in 20 mM phosphate buffer, pH 6.5. A column of Navy HE-R adsorbent was used first to remove most of the potentially adsorbing proteins. PMID:6326622

  14. Improved method for the on-line metal chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products.

    PubMed

    Cooper, A D; Stubbings, G W; Kelly, M; Tarbin, J A; Farrington, W H; Shearer, G

    1998-07-01

    An improved on-line metal chelate affinity chromatography-high-performance liquid chromatography (MCAC-HPLC) method for the determination of tetracycline antibiotics in animal tissues and egg has been developed. Extraction was carried out with ethyl acetate. The extract was then evaporated to dryness and reconstituted in methanol prior to on-line MCAC clean-up and HPLC-UV determination. Recoveries of tetracycline, oxytetracycline, demeclocycline and chlortetracycline in the range 42% to 101% were obtained from egg, poultry, fish and venison tissues spiked at 25 micrograms kg-1. Limits of detection less than 10 microgram kg-1 were estimated for all four analytes. This method has higher throughput, higher recovery and lower limits of detection than a previously reported on-line MCAC-HPLC method which involved aqueous extraction and solid-phase extraction clean-up. PMID:9691328

  15. Stereoselective Binding of Chiral Ligands to Single Nucleotide Polymorphs (SNPs) of the Human Organic Cation Transporter-1 Determined Using Cellular Membrane Affinity Chromatography

    PubMed Central

    Moaddel, R.; Bighi, F.; Yamaguchi, R.; Patel, S.; Ravichandran, S.; Wainer, I.W.

    2010-01-01

    Membranes from stably transfected cell lines that expresses two point mutations of the human organic cation 1 transporter (hOCT1), R488M and G465R, have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the Cellular Membrane Affinity Chromatography (CMAC) (hOCT1G465R) and CMAC(hOCT1R488M). Columns were created using both stationary phases and frontal displacement chromatography experiments were conducted using [3H]-methyl phenyl pyridinium, [3H]-MPP+, as the marker ligand and various displacers, including the single enantiomers of verapamil, fenoterol and isoproterenol. The chromatographic data obtained was used to refine a previously developed pharmacophore for the hOCT1 transporter. PMID:20206116

  16. Analysis of drug-protein interactions by high-performance affinity chromatography: interactions of sulfonylurea drugs with normal and glycated human serum albumin.

    PubMed

    Matsuda, Ryan; Anguizola, Jeanethe; Hoy, Krina S; Hage, David S

    2015-01-01

    High-performance affinity chromatography (HPAC) is a type of liquid chromatography that has seen growing use as a tool for the study of drug-protein interactions. This report describes how HPAC can be used to provide information on the number of binding sites, equilibrium constants, and changes in binding that can occur during drug-protein interactions. This approach will be illustrated through recent data that have been obtained by HPAC for the binding of sulfonylurea drugs and other solutes to the protein human serum albumin (HSA), and especially to forms of this protein that have been modified by non-enzymatic glycation. The theory and use of both frontal analysis and zonal elution competition studies in such work will be discussed. Various practical aspects of these experiments will be presented, as well as factors to consider in the extension of these methods to other drugs and proteins or additional types of biological interactions. PMID:25749961

  17. Soybean Cyst Nematode SDS-PAGE Protein Characterization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) represent one of the most serious threats to the stability of soybean crops in the United States. Initially discovered in North Carolina in the 1950s, it has spread rapidly through the Midwest generating an estimated $1 billion in failed crop...

  18. Chromatography

    MedlinePlus

    ... a way of separating two or more chemical compounds. Chemical compounds are chemicals that are bonded together. For example, ... and hydrogen. Proteins are another type of chemical compound. There are different kinds of chromatography. These include ...

  19. Surface plasmon resonance spectroscopy-based high-throughput screening of ligands for use in affinity and displacement chromatography.

    PubMed

    Vutukuru, Srinavya; Kane, Ravi S

    2008-10-21

    We describe an approach that uses surface plasmon resonance (SPR) spectroscopy and self-assembled monolayers (SAMs) for the high-throughput screening of ligands for use in displacement and affinity chromatographic processes. We identified a set of commercially available organic amines and allowed them to react with SAMs presenting interchain carboxylic anhydride groups; the resulting surfaces presented ligands of interest in a background of carboxylic acid groups. We used SPR spectroscopy to determine the extent of adsorption of two model proteinslysozyme and cytochrome conto these "multimodal" surfaces and to select promising "affinity" ligands for further characterization. The attachment of selected ligands to UltraLink Biosupport resulted in beads with a significantly greater affinity for lysozyme than for cytochrome c that would be suitable for use in affinity chromatographic processes. Furthermore, we also used the screens to design "affinity displacers"small molecules that selectively retain lysozyme on chromatographic resins, while displacing cytochrome c. The combination of SPR spectroscopy and SAMs represents a powerful technique for identifying novel ligands that enable the purification of complex protein mixtures. PMID:18788766

  20. Preparation of a high-performance multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human plasma glycoproteins.

    PubMed

    Kullolli, Majlinda; Hancock, William S; Hincapie, Marina

    2008-08-01

    We report on the preparation of an improved multi-lectin affinity support for HPLC separations. We combined the selectivity of three different lectins: concanavalin A (ConA), wheat germ agglutinin (WGA), and jacalin (JAC). Each lectin was first covalently immobilized onto a polymeric matrix and then the three lectin media were combined in equal ratios. The beads were packed into a column to produce a mixed-bed multi-lectin HPLC column (high-performance multi-lectin affinity chromatography (HP-M-LAC)) for fast chromatographic affinity separations. The support was characterized with respect to kinetics of immobilization, ligand density, and binding capacity for human plasma glycoproteins. A high lectin density (15 mg/mL of beads) was found to be optimal for the binding of glycoproteins from human plasma. A single clinical sample can be fractionated in less than 10 min per run, making this a useful sample preparation tool for proteomics/glycoproteomics studies associated with disease abnormalities. PMID:18693314

  1. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

    PubMed

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S

    2016-08-01

    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent. PMID:27289464

  2. Affinity chromatography, two-dimensional electrophoresis, adapted immunodepletion and mass spectrometry used for detection of porcine and piscine heparin-binding human plasma proteins.

    PubMed

    Bjarnadóttir, Stefanía Guðrún; Flengsrud, Ragnar

    2014-01-01

    Heparin-binding proteins in human plasma were studied using affinity chromatography columns with porcine (2mL, 10.7mg capacity) and piscine heparin (5mL, 2.7mg capacity). Two-dimensional electrophoresis (Bio-Rad Protean II gel system with 16cm×16cm gels using isoelectric focusing (IEF) and nonequilibrium pH-gradient gel electrophoresis (NEPHGE)), Bruker Ultraflex MALDI-TOF mass spectrometry and immunoblotting (NovaBlot semidry discontinuous blotting) were used for unfractionated plasma. This revealed electropherograms with differences between porcine and piscine heparin-binding and totally 17 different fibrinogen variants from all 3 chains. Immunodepletion was used to remove fibrinogen (42.1mg anti-human fibrinogen in 8.4mL resin) and serum albumin (0.42mg binding capacity in 14mL resin) and porcine and piscine heparin-binding proteins were identified using liquid chromatography-mass spectrometry (Ultimate 3000 NanoLC with Acclaim PepMap 100 column (50cm×75μm)-LTQ Orbitrap Mass XL). In total, the binding of 76 putative or acknowledged biomarkers are shown. Of the identified proteins, 14 are not previously shown to be heparin-binding, such as the low concentration proteins lipocalin-1 and tropomyosin and a hitherto not detected protein in plasma, zinc finger protein 483. The putative heparin-binding sequences were analyzed. The results suggest that the combination of group specific affinity and adapted immunodepletion chromatography could be useful in the study of the plasma proteome. PMID:24316520

  3. Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

    PubMed Central

    Jackson, Abby J.; Anguizola, Jeanethe; Pfaunmiller, Erika L.; Hage, David S.

    2013-01-01

    Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1 × 104–3 × 105 M−1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions. PMID:23657448

  4. One-step purification of glucoamylase by affinity precipitation with alginate.

    PubMed

    Teotia, S; Lata, R; Khare, S K; Gupta, M N

    2001-01-01

    It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens. PMID:11746949

  5. Hydrophilic polydopamine-coated graphene for metal ion immobilization as a novel immobilized metal ion affinity chromatography platform for phosphoproteome analysis.

    PubMed

    Yan, Yinghua; Zheng, Zhifang; Deng, Chunhui; Li, Yan; Zhang, Xiangmin; Yang, Pengyuan

    2013-09-17

    To discover trace phosphorylated proteins or peptides with great biological significance for in-depth phosphoproteome analysis, it is urgent to develop a novel technique for highly selective and effective enrichment of phosphopeptides. In this work, an IMAC (immobilized metal ion affinity chromatography) material with polydopamine coated on the surface of graphene and functionalized with titanium ions (denoted as Ti(4+)-G@PD) was initially designed and synthesized. The newly prepared Ti(4+)-G@PD with enhanced hydrophilicity and biological compatibility was characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and infrared (IR), and its performance for selective and effective enrichment of phosphopeptide was evaluated with both standard peptide mixtures and human serum. PMID:23941301

  6. Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

    PubMed Central

    Zlatic, Stephanie A.; Ryder, Pearl V.; Salazar, Gloria; Faundez, Victor

    2010-01-01

    The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 Å, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1. Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work. PMID:20216526

  7. Synthesis and characterization of a cellular membrane affinity chromatography column containing histamine 1 and P2Y1 receptors: A multiple G-protein coupled receptor column

    PubMed Central

    Moaddel, Ruin; Musyimi, Harrison K.; Sanghvi, Mitesh; Bashore, Charlene; Frazier, Chester R.; Khadeer, Mohammad; Bhatia, Prateek; Wainer, Irving W.

    2015-01-01

    A cellular membrane affinity chromatography (CMAC) column has been created using cellular membrane fragments from a 1321N1 cell line stably transfected with the P2Y1 receptor. The CMAC(1321N1P2Y1) column contained functional P2Y1 and histamine 1 receptors, which independently bound receptor-specific ligands. The data obtained with the CMAC(1321N1P2Y1) column demonstrate that multiple-G-protein coupled receptor (GPCR) columns can be developed and used to probe interactions with the immobilized receptors and that endogenously expressed GPCRs can be used to create CMAC columns. The results also establish that the histamine 1 receptor can be immobilized with retention of ligand-specific binding. PMID:19608372

  8. Use of a Phosphatidylinositol Phosphate Affinity Chromatography (PIP Chromatography) for the Isolation of Proteins Involved in Protein Quality Control and Proteostasis Mechanisms in Plants.

    PubMed

    Farmaki, T

    2016-01-01

    Protein functionality depends directly on its accurately defined three-dimensional organization, correct and efficient posttranslational modification, and transport. However, proteins are continuously under a hostile environment threatening with folding aberrations, aggregation, and mistargeting. Therefore, proteins must be constantly "followed up" by a tightly regulated homeostatic mechanism specifically known as proteostasis. To this end other proteins ensure this close surveillance including chaperones as well as structural and functional members of the proteolytic mechanisms, mainly the autophagy and the proteasome related. They accomplish their action via interactions not only with other proteins but also with lipids as well as cytoskeletal components. We describe a protocol based on an affinity chromatographic approach aiming at the isolation of phosphatidyl inositol phosphate binding proteins, a procedure which results into the enrichment and purification of several members of the proteostasis mechanism, e.g. autophagy and proteasome, among other components of the cell signaling pathways. PMID:27424758

  9. Sialic acid-specific affinity chromatography for the separation of erythropoietin glycoforms using serotonin as a ligand.

    PubMed

    Meininger, M; Stepath, M; Hennig, R; Cajic, S; Rapp, E; Rotering, H; Wolff, M W; Reichl, U

    2016-02-15

    Recombinant human erythropoietin (rhEPO) is an important CHO cell-derived glycoprotein and the degree of sialylation of this hormone is crucial for its in vivo bioactivity. In order to improve the purification process serotonin as a potential affinity ligand was tested for preparative chromatographic separation of rhEPO glycoforms into fractions of different degrees of sialylation. Therefore, two chromatographic matrices were prepared by immobilizing serotonin on CNBr- and NHS-Sepharose™. First it was shown both matrices bind rhEPO only in its sialylated form. Results indicate that binding is pH independent between pH 3.5 to 8 suggesting it is not only based on electrostatic interactions. Second, after optimal binding conditions were identified, semi-purified rhEPO was loaded onto both matrices and eluted using a stepwise elution gradient of sodium chloride. For comparison same affinity purification experiments were performed using wheat germ agglutinin-coupled agarose, a lectin known for its affinity towards sialylated glycoproteins. To monitor changes in N-glycan fingerprint, eluate fractions were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence (xCGE-LIF). For the serotonin matrices an increasing degree of sialylation was observed from the first to the third elution fraction while purity of rhEPO could be increased at the same time. The late elution fractions of serotonin-coupled CNBr- and NHS-Sepharose™ also showed an overall sialylation degree exceeding that of the starting material. In contrast, for rhEPO bound to wheat germ agglutinin-coupled agarose, no distinct change in the degree of sialylation could be observed after elution. Overall, these encouraging results highlight the potential of serotonin as a chromatographic ligand for the improvement of pharmaceutical purification processes of rhEPO. PMID:26851523

  10. Enhanced DR5 binding capacity of nanovectorized TRAIL compared to its cytotoxic version by affinity chromatography and molecular docking studies.

    PubMed

    Zakaria, Albatoul; Picaud, Fabien; Guillaume, Yves Claude; Gharbi, Tijani; Micheau, Olivier; Herlem, Guillaume

    2016-09-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of cancer cells when bound to its cognate receptors, TRAIL-R1 and TRAIL-R2 (DR4 and DR5), without being toxic to healthy cells. Nanovectorized TRAIL (abbreviated as NPT) is 10 to 20 times more efficient than one of the most potent soluble TRAIL used in preclinical studies (His-TRAIL). To determine whether differences in affinity may account for NPT superiority, a thermodynamic study was undertaken to evaluate NPT versus TRAIL binding affinity to DR5. Docking calculations showed that TRAIL in homotrimer configuration was more stable than in heterotrimer, because of the presence of one Zn ion in its structure. Indeed, TRAIL trimers can have head-to-tail orientations when Zn is missing. Altogether these data suggest that TRAIL homotrimer structures are predominant in solution and then are grafted on NPT. When docked to DR5, NPT carrying TRAIL homotrimer leads to a more stable complex than TRAIL monomer-based NPT. To comfort these observations, the extracellular domain of DR5 was immobilized on a chromatographic support using an "in situ" immobilization technique. The determination of the thermodynamic data (enthalpy ∆H° and entropy ∆S°*) of TRAIL and NPT binding to DR5 showed that the binding mechanism was pH dependent. The affinity of NPT to DR5 increased with pH, and the ionized energy was more important for NPT than for soluble TRAIL. Moreover, because of negative values of ∆H° and ∆S°* quantities, we demonstrated that van der Waals and hydrogen bonds governed the strong NPT-DR5 association for pH > 7.4 (as for TRAIL alone). Copyright © 2016 John Wiley & Sons, Ltd. PMID:26952193

  11. EVALUATION OF ALTERNATIVES TO WARFARIN AS PROBES FOR SUDLOW SITE I OF HUMAN SERUM ALBUMIN CHARACTERIZATION BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Joseph, K.S.; Moser, Annette C.; Basiaga, Sara; Schiel, John E.; Hage, David S.

    2009-01-01

    Warfarin is often used as a site-specific probe for examining the binding of drugs and other solutes to Sudlow site I of human serum albumin (HSA). However, warfarin has strong binding to HSA and the two chiral forms of warfarin have slightly different binding affinities for this protein. Warfarin also undergoes a slow change in structure when present in common buffers used for binding studies. This report examined the use of four related, achiral compounds (i.e., coumarin, 7-hydroxycoumarin, 7-hydroxy-4-methylcoumarin, and 4-hydroxycoumarin) as possible alternative probes for Sudlow site I in drug binding studies. High-performance affinity chromatography and immobilized HSA columns were used to compare and evaluate the binding properties of these probe candidates. Binding for each of the tested probe candidates to HSA was found to give a good fit to a two-site model. The first group of sites had moderate-to-high affinities for the probe candidates with association equilibrium constants that ranged from 6.4 × 103 M−1 (coumarin) to 5.5 × 104 M−1 (4-hydroxycoumarin) at pH 7.4 and 37°C. The second group of weaker, and probably non-specific, binding regions, had association equilibrium constants that ranged from 3.8 × 101 M−1 (7-hydroxy-4-methylcoumarin) to 7.3 × 102 M−1 (coumarin). Competition experiments based on zonal elution indicated that all of these probe candidates competed with warfarin at their high affinity regions. Warfarin also showed competition with coumarin, 7-hydroxycoumarin and 7-hydroxy-4-methycoumarin for their weak affinity sites but appeared to not bind and or compete for all of the weak sites of 4-hydroxycoumarin. It was found from this group that 4-hydroxycoumarin was the best alternative to warfarin for examining the interactions of drugs at Sudlow site I on HSA. These results also provided information on how the major structural components of warfarin contribute to the binding of this drug at Sudlow site I. PMID:18926542

  12. Characterization of biases in phosphopeptide enrichment by Ti(4+)-immobilized metal affinity chromatography and TiO2 using a massive synthetic library and human cell digests.

    PubMed

    Matheron, Lucrece; van den Toorn, Henk; Heck, Albert J R; Mohammed, Shabaz

    2014-08-19

    Outcomes of comparative evaluations of enrichment methods for phosphopeptides depend highly on the experimental protocols used, the operator, the source of the affinity matrix, and the samples analyzed. Here, we attempt such a comparative study exploring a very large synthetic library containing thousands of serine, threonine, and tyrosine phosphorylated peptides, being present in roughly equal abundance, along with their nonphosphorylated counterparts, and use an optimized protocol for enrichment by TiO2 and Ti(4+)-immobilized metal affinity chromatography (IMAC) by a single operator. Surprisingly, our data reveal that there are minimal differences between enrichment of phosphopeptides by TiO2 and Ti(4+)-IMAC when considering biochemical and biophysical parameters such as peptide length, sequence surrounding the site, hydrophobicity, and nature of the amino acid phosphorylated. Similar results were obtained when evaluating a tryptic digest of a cellular lysate, representing a more natural source of phosphopeptides. All the data presented are available via ProteomeXchange with the identifier PXD000759. PMID:25068997

  13. Online micro-solid-phase extraction based on boronate affinity monolithic column coupled with high-performance liquid chromatography for the determination of monoamine neurotransmitters in human urine.

    PubMed

    Yang, Xiaoting; Hu, Yufei; Li, Gongke

    2014-05-16

    Quantification of monoamine neurotransmitters is very important in diagnosing and monitoring of patients with neurological disorders. We developed an online analytical method to selectively determine urinary monoamine neurotransmitters, which coupled the boronate affinity monolithic column micro-solid-phase extraction with high-performance liquid chromatography (HPLC). The boronate affinity monolithic column was prepared by in situ polymerization of vinylphenylboronic acid (VPBA) and N,N'-methylenebisacrylamide (MBAA) in a stainless capillary column. The prepared monolithic column showed good permeability, high extraction selectivity and capacity. The column-to-column reproducibility was satisfactory and the enrichment factors were 17-243 for four monoamine neurotransmitters. Parameters that influence the online extraction efficiency, including pH of sample solution, flow rate of extraction and desorption, extraction volume and desorption volume were investigated. Under the optimized conditions, the developed method exhibited low limit of detection (0.06-0.80μg/L), good linearity (with R(2) between 0.9979 and 0.9993). The recoveries in urine samples were 81.0-105.5% for four monoamine neurotransmitters with intra- and inter-day RSDs of 2.1-8.2% and 3.7-10.6%, respectively. The online analytical method was sensitive, accurate, selective, reliable and applicable to analysis of trace monoamine neurotransmitters in human urine sample. PMID:24703360

  14. Preliminary study of the metal binding site of an anti-DTPA-indium antibody by equilibrium binding immunoassays and immobilized metal ion affinity chromatography.

    PubMed

    Boden, V; Colin, C; Barbet, J; Le Doussal, J M; Vijayalakshmi, M

    1995-01-01

    Creating metal coordination sites by modifying an existing enzyme or by eliciting antibodies against metal chelate haptens is of great interest in biotechnology to create enzyme catalysts with novel specificities. Here, we investigate the metal binding potential of a monoclonal antibody raised against a DTPA-In(III) hapten (mAb 734). We study its relative binding efficiency to metals of biological relevance by equilibrium binding immunoassays and immobilized metal ion affinity chromatography, two approaches which can give complementary information regarding composition and/or structure of the metal binding site(s). Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(II) binding was compared to In(III). All of them were shown to displace indium, but their affinity for mAb 734 decreased by 100-fold compared to indium. Competitive metal binding immunoassays between Zn(II) and In(III) revealed an unusual behavior by Zn(II) which remains to be explained. Moreover, IMAC allowed us to predict the metal binding amino acids involved in the antibody paratope. The antibody metal binding site was shown to contain at least two histidine residues in a cluster, and the presence of aspartic and glutamic acid as well as cysteine residues could not be excluded. Thus, simple competition studies allows us to obtain some partial information on the metal binding structural features of this anti-metal chelate antibody and to guide our screening of its catalytic potential. PMID:7578356

  15. Analytical high-performance affinity chromatography: evaluation by studies of neurophysin self-association and neurophysin-peptide hormone interaction using glass matrices

    SciTech Connect

    Swaisgood, H.E.; Chaiken, I.M.

    1986-07-01

    Bovine neurophysin II (BNP II) was covalently immobilized on both nonporous and porous (200-nm pore diameter) glass beads and incorporated in a high-performance liquid chromatograph to evaluate analytical high-performance affinity chromatography as a microscale method for characterizing biomolecular interactions. The self-association of neurophysin and its binding of the peptide hormone vasopressin were characterized by using a single chromatograhic column containing immobilized neurophysin predominantly in the monomer form. Both (/sup 3/H)(Arg/sup 8/)vasopressin (AVP) and /sup 125/I-BNP II were rapidly eluted (<25 min). The relatively symmetrical elution peaks obtained allowed calculation of both equilibrium dissociation constants and kinetic dissociation rate constants. In contrast to the agreement of chromatographic equilibrium binding constants with those measured in solution, the dissociation rate, k..sqrt../sub 3/, determined from the variance of the affinity chromatographic elution profile with nonporous beads, was several orders of magnitude smaller than the solution counterpart. This latter difference may reflect the probability of rebinding to contiguous sites immobilized on a surface, a feature which would be related to that for contiguous sites on a membrane.

  16. A proteomics platform combining depletion, multi-lectin affinity chromatography (M-LAC), and isoelectric focusing to study the breast cancer proteome.

    PubMed

    Zeng, Zhi; Hincapie, Marina; Pitteri, Sharon J; Hanash, Samir; Schalkwijk, Joost; Hogan, Jason M; Wang, Hong; Hancock, William S

    2011-06-15

    The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation, and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as to simultaneously detect glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation, and LC-MS analysis has been applied to discover breast cancer-associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component, and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies. PMID:21513341

  17. A Proteomics Platform Combining Depletion, Multi-lectin Affinity Chromatography (M-LAC) and Isoelectric Focusing to Study the Breast Cancer Proteome

    PubMed Central

    Zeng, Zhi; Hincapie, Marina; Pitteri, Sharon J.; Hanash, Samir; Schalkwijk, Joost; Hogan, Jason M.; Wang, Hon; Hancock, William S.

    2011-01-01

    The discovery of breast cancer associated plasma/serum biomarkers is important for early diagnosis, disease mechanism elucidation and determination of treatment strategy for the disease. In this study of serum samples, a multidimensional fractionation platform combined with mass spectrometric analysis were used to achieve the identification of medium to lower abundance proteins, as well as simultaneously detecting glycan and abundance changes. Immuno-affinity depletion and multi-lectin chromatography (M-LAC) were integrated into an automated HPLC platform to remove high abundance protein and fractionate glycoproteins. The collected glycoproteomes were then subjected to isoelectric focusing (IEF) separation by a digital ProteomeChip (dPC), followed by in-gel digestion and LC-MS analysis using an Orbitrap mass spectrometer. As a result, the total number of identified proteins increased significantly when the IEF fractionation step was included as part of the platform. Relevant proteins with biological and disease significance were observed and the dynamic range of the serum proteome measurement was extended. In addition, potential glycan changes were indicated by comparing proteins in control and cancer samples in terms of their affinity to the multi-lectin column (M-LAC) and the pI profiles in IEF separation. In conclusion, a proteomics platform including high abundance protein depletion, lectin affinity fractionation, IEF separation and LC-MS analysis has been applied to discover breast cancer associated proteins. The following candidates, thrombospondin-1 and 5, alpha-1B-glycoprotein, serum amyloid P-component and tenascin-X, were selected as promising examples of the use of this platform. They show potential abundance and glycan changes and will be further investigated in future studies. PMID:21513341

  18. Nanoengineered analytical immobilized metal affinity chromatography stationary phase by atom transfer radical polymerization: Separation of synthetic prion peptides

    PubMed Central

    McCarthy, P.; Chattopadhyay, M.; Millhauser, G.L.; Tsarevsky, N.V.; Bombalski, L.; Matyjaszewski, K.; Shimmin, D.; Avdalovic, N.; Pohl, C.

    2010-01-01

    Atom transfer radical polymerization (ATRP) was employed to create isolated, metal-containing nanoparticles on the surface of non-porous polymeric beads with the goal of developing a new immobilized metal affnity chromatography (IMAC) stationary phase for separating prion peptides and proteins. Transmission electron microscopy was used to visualize nanoparticles on the substrate surface. Individual ferritin molecules were also visualized as ferritin–nanoparticle complexes. The column's resolving power was tested by synthesizing peptide analogs to the copper binding region of prion protein and injecting mixtures of these analogs onto the column. As expected, the column was capable of separating prion-related peptides differing in number of octapeptide repeat units (PHGGGWGQ), (PHGGGWGQ)2, and (PHGGGWGQ)4. Unexpectedly, the column could also resolve peptides containing the same number of repeats but differing only in the presence of a hydrophilic tail, Q → A substitution, or amide nitrogen methylation. PMID:17481564

  19. Evaluation of microbeads of calcium alginate as a fluidized bed medium for affinity chromatography of Aspergillus niger Pectinase.

    PubMed

    Roy, Ipsita; Jain, Sulakshana; Teotia, Sunita; Gupta, Munishwar Nath

    2004-01-01

    Calcium alginate microbeads (212-425 microm) were prepared by spraying 2% (w/v) alginate solution into 1 M CaCl2 solution. The fluidization behavior of these beads was studied, and the bed expansion index and terminal velocity were found to be 4.3 and 1808 cm h(-1), respectively. Residence time distribution curves showed that the dispersion of the protein was much less with these microbeads than with conventionally prepared calcium alginate macrobeads when both kinds of beads were used for chromatography in a fluidized bed format. The fluidized bed of these beads was used for the purification of pectinase from a commercial preparation. The media performed well even with diluted feedstock; 90% activity recovery with 211-fold purification was observed. PMID:15458334

  20. The isolation by ligand affinity chromatography of a novel form of alpha-L-fucosidase from almond.

    PubMed

    Scudder, P; Neville, D C; Butters, T D; Fleet, G W; Dwek, R A; Rademacher, T W; Jacob, G S

    1990-09-25

    An alpha-fucosidase has been extracted from almond meal and purified 163,000-fold to apparent homogeneity using a novel affinity ligand, N-(5-carboxy-1-pentyl)-1,5-dideoxy-1,5-imino-L-fucitol, coupled to Affi-Gel 102. Substrate specificity studies demonstrate that the enzyme hydrolyzes the alpha-fucosidic linkages in Gal(beta 1----3)(Fuc(alpha 1----4]GlcNAc(beta 1----3)Gal(beta 1----4)Glc and Gal(beta 1----4)(Fuc(alpha 1----3]GlcNAc(beta 1----3)Gal(beta 1----4)Glc at similar rates but is unable to hydrolyze Fuc(alpha 1----2)Gal, Fuc(alpha 1----6)GlcNAc, or the synthetic substrate, p-nitrophenyl alpha-L-fucopyranoside. Hence, the enzyme closely resembles an alpha-fucosidase I isolated previously from a commercial preparation of partially purified almond beta-glucosidase (Ogata-Arakawa, M., Muramatsu, T., and Kobata, A. (1977) Arch. Biochem. Biophys. 181, 353-358). However, native and subunit relative molecular masses of 106,000 and 54,000 respectively, different charge and hydrophobicity properties, and the absence of stimulation by NaCl clearly distinguish this enzyme, designated alpha-fucosidase III, from other almond alpha-fucosidases reported previously. PMID:2398059

  1. Rapid Screening of Drug-Protein Binding Using High-Performance Affinity Chromatography with Columns Containing Immobilized Human Serum Albumin

    PubMed Central

    Li, Ying-Fei; Zhang, Xiao-Qiong; Hu, Wei-Yu; Li, Zheng; Liu, Ping-Xia; Zhang, Zhen-Qing

    2013-01-01

    For drug candidates, a plasma protein binding (PPB) more than 90% is more meaningful and deserves further investigation in development. In the study, a high-performance liquid chromatography method employing column containing immobilized human serum albumin (HSA) to screen in vitro PPB of leading compounds was established and successfully applied to tested compounds. Good correlation (a coefficient correlation of 0.96) was attained between the reciprocal values (X) of experimentally obtained retention time of reference compounds eluted through HSA column and the reported PPB values (Y) with a correlation equation of Y = 92.03 − 97.01X. The method was successfully applied to six test compounds, and the result was confirmed by the conventional ultrafiltration technique, and both yielded equal results. However, due to the particular protein immobilized to column, the method cannot be applied for all compounds and should be exploited judiciously based on the value of the logarithmic measure of the acid dissociation constant (pKa) as per the requirement. If α1-acid glycoprotein and other plasma proteins could be immobilized like HSA with their actual ratio in plasma to column simultaneously, the result attained using immobilized column may be more accurate, and the method could be applied to more compounds without pKa limitation. PMID:23607050

  2. Study of immobilized metal affinity chromatography sorbents for the analysis of peptides by on-line solid-phase extraction capillary electrophoresis-mass spectrometry.

    PubMed

    Ortiz-Martin, Lorena; Benavente, Fernando; Medina-Casanellas, Silvia; Giménez, Estela; Sanz-Nebot, Victoria

    2015-03-01

    Several commercial immobilized metal affinity chromatography sorbents were evaluated in this study for the analysis of two small peptide fragments of the amyloid β-protein (Aβ) (Aβ(1-15) and Aβ(10-20) peptides) by on-line immobilized metal affinity SPE-CE (IMA-SPE-CE). The performance of a nickel metal ion (Ni(II)) sorbent based on nitrilotriacetic acid as a chelating agent was significantly better than two copper metal ion (Cu(II)) sorbents based on iminodiacetic acid. A BGE of 25 mM phosphate (pH 7.4) and an eluent of 50 mM imidazole (in BGE) yielded a 25-fold and 5-fold decrease in the LODs by IMA-SPE-CE-UV for Aβ(1-15) and Aβ(10-20) peptides (0.1 and 0.5 μg/mL, respectively) with regard to CE-UV (2.5 μg/mL for both peptides). The phosphate BGE was also used in IMA-SPE-CE-MS, but the eluent needed to be substituted by a 0.5% HAc v/v solution. Under optimum preconcentration and detection conditions, reproducibility of peak areas and migration times was acceptable (23.2 and 12.0%RSD, respectively). The method was more sensitive for Aβ(10-20) peptide, which could be detected until 0.25 μg/mL. Linearity for Aβ(10-20) peptide was good in a narrow concentration range (0.25-2.5 μg/mL, R(2) = 0.93). Lastly, the potential of the optimized Ni(II)-IMA-SPE-CE-MS method for the analysis of amyloid peptides in biological fluids was evaluated by analyzing spiked plasma and serum samples. PMID:25640944

  3. Simple Method for Shiga Toxin 2e Purification by Affinity Chromatography via Binding to the Divinyl Sulfone Group

    PubMed Central

    Arimitsu, Hideyuki; Sasaki, Keiko; Kojima, Hiroe; Yanaka, Tadashi; Tsuji, Takao

    2013-01-01

    Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease. PMID:24340102

  4. Efficient and rapid purification of lentil alpha-galactosidase by affinity precipitation with alginate.

    PubMed

    Celem, Evran Biçak; Bolle, Sharon Sibel; Onal, Seçil

    2009-10-01

    Alpha-Galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was purified (26-fold) from the germinating seeds of lentil (Lens culinaris) by affinity precipitation with alginate. The purified enzyme gave a single band corresponding to molecular mass of 40 kDa on SDS-PAGE. The optimum temperature and pH of the enzyme were determined as 40 degrees C and 5.5, respectively. The enzyme was very stable at a temperature range of 4-65 degrees C and at a pH range of 4-7. The values of kinetic constants Km and Vmax using p-nitrophenyl-alpha-D-galactopyranoside (PNPG) as substrate were 0.191 mM and 0.73 U, respectively. Results suggest that affinity precipitation is an attractive process for the purification of alpha-galactosidase. PMID:20027865

  5. Quantitative Phosphoproteome Analysis of Lysophosphatidic Acid Induced Chemotaxis applying Dual-step ¹⁸O Labeling Coupled with Immobilized Metal-ion Affinity Chromatography

    SciTech Connect

    Ding, Shi-Jian; Wang, Yingchun; Jacobs, Jon M.; Qian, Weijun; Yang, Feng; Tolmachev, Aleksey V.; Du, Xiuxia; Wang, Wei; Moore, Ronald J.; Monroe, Matthew E.; Purvine, Samuel O.; Waters, Katrina M.; Heibeck, Tyler H.; Adkins, Joshua N.; Camp, David G.; Klemke, Richard L.; Smith, Richard D.

    2008-10-01

    Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in a variety of different cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its applications for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed 16O/18O labeling plus 16O/18O-methanol esterification labeling for quantitation, a macro- Immobilized Metal-ion Affinity Chromatography trap for phosphopeptide enrichment, and a monolithic capillary column with integrated electrospray emitter. LC separation and MS/MS is followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer and complementary searching algorithms for interpreting MS/MS spectra. Protein phosphorylation involved in various signaling pathways of cell migration were identified and quantified, such as mitogen-activated protein kinase 1, dual-specificity mitogen-activated protein kinase kinase 2, and dual-specificity tyrosine-phosphorylation regulated kinase 1b, and a number of Rho GTPase-activating proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with gradient sensing and cell chemotaxis.

  6. Identification of phosphoproteins in Arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metal-ion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Aryal, Uma K; Krochko, Joan E; Ross, Andrew R S

    2012-01-01

    Reversible protein phosphorylation is a key regulatory mechanism in cells. Identification and characterization of phosphoproteins requires specialized enrichment methods, due to the relatively low abundance of these proteins, and is further complicated in plants by the high abundance of Rubisco in green tissues. We present a novel method for plant phosphoproteome analysis that depletes Rubisco using polyethylene glycol fractionation and utilizes immobilized metal-ion affinity chromatography to enrich phosphoproteins. Subsequent protein separation by one- and two-dimensional gel electrophoresis is further improved by extracting the PEG-fractionated protein samples with SDS/phenol and methanol/chloroform to remove interfering compounds. Using this approach, we identified 132 phosphorylated proteins in a partial Arabidopsis leaf extract. These proteins are involved in a range of biological processes, including CO(2) fixation, protein assembly and folding, stress response, redox regulation, and cellular metabolism. Both large and small subunits of Rubisco were phosphorylated at multiple sites, and depletion of Rubisco enhanced detection of less abundant phosphoproteins, including those associated with state transitions between photosystems I and II. The discovery of a phosphorylated form of AtGRP7, a self-regulating RNA-binding protein that affects floral transition, as well as several previously uncharacterized ribosomal proteins confirm the utility of this approach for phosphoproteome analysis and its potential to increase our understanding of growth and development in plants. PMID:22092075

  7. An in depth proteomic analysis based on ProteoMiner, affinity chromatography and nano-HPLC-MS/MS to explain the potential health benefits of bovine colostrum.

    PubMed

    Altomare, Alessandra; Fasoli, Elisa; Colzani, Mara; Parra, Ximena Maria Paredes; Ferrari, Marina; Cilurzo, Francesco; Rumio, Cristiano; Cannizzaro, Luca; Carini, Marina; Righetti, Pier Giorgio; Aldini, Giancarlo

    2016-03-20

    Bovine colostrum (BC), the initial milk secreted by the mammary gland immediately after parturition, is widely used for several health applications. We here propose an off-target method based on proteomic analysis to explain at molecular level the potential health benefits of BC. The method is based on the set-up of an exhaustive protein data bank of bovine colostrum, including the minor protein components, followed by a bioinformatic functional analysis. The proteomic approach based on ProteoMiner technology combined to a highly selective affinity chromatography approach for the immunoglobulins depletion, identified 1786 proteins (medium confidence; 634 when setting high confidence), which were then clustered on the basis of their biological function. Protein networks were then created on the basis of the biological functions or health claims as input. A set of 93 proteins involved in the wound healing process was identified. Such an approach also permits the exploration of novel biological functions of BC by searching in the database the presence of proteins characterized by innovative functions. In conclusion an advanced approach based on an in depth proteomic analysis is reported which permits an explanation of the wound healing effect of bovine colostrum at molecular level and allows the search of novel potential beneficial effects. PMID:26809613

  8. Penetrable silica microspheres for immobilization of bovine serum albumin and their application to the study of the interaction between imatinib mesylate and protein by frontal affinity chromatography.

    PubMed

    Ma, Liyun; Li, Jing; Zhao, Juan; Liao, Han; Xu, Li; Shi, Zhi-guo

    2016-01-01

    In the current study, novel featured silica, named penetrable silica, simultaneously containing macropores and mesopores, was immobilized with bovine serum albumin (BSA) via Schiff base method. The obtained BSA-SiO2 was employed as the high-performance liquid chromatographic (HPLC) stationary phase. Firstly, D- and L-tryptophan were used as probes to investigate the chiral separation ability of the BSA-SiO2 stationary phase. An excellent enantioseparation factor was obtained up to 4.3 with acceptable stability within at least 1 month. Next, the BSA-SiO2 stationary phase was applied to study the interaction between imatinib mesylate (IM) and BSA by frontal affinity chromatography. A single type of binding site was found for IM with the immobilized BSA, and the hydrogen-bonding and van der Waals interactions were expected to be contributing interactions based on the thermodynamic studies, and this was a spontaneous process. Compared to the traditional silica for HPLC stationary phase, the proposed penetrable silica microsphere possessed a larger capacity to bond more BSA, minimizing column overloading effects and enhancing enantioseparation ability. In addition, the lower running column back pressure and fast mass transfer were meaningful for the column stability and lifetime. It was a good substrate to immobilize biomolecules for fast chiral resolution and screening drug-protein interactions. PMID:26573171

  9. Immobilized metal affinity chromatography on collapsed Langmuir-Blodgett iron(III) stearate films and iron(III) oxide nanoparticles for bottom-up phosphoproteomics.

    PubMed

    Gladilovich, Vladimir; Greifenhagen, Uta; Sukhodolov, Nikolai; Selyutin, Artem; Singer, David; Thieme, Domenika; Majovsky, Petra; Shirkin, Alexey; Hoehenwarter, Wolfgang; Bonitenko, Evgeny; Podolskaya, Ekaterina; Frolov, Andrej

    2016-04-22

    Phosphorylation is the enzymatic reaction of site-specific phosphate transfer from energy-rich donors to the side chains of serine, threonine, tyrosine, and histidine residues in proteins. In living cells, reversible phosphorylation underlies a universal mechanism of intracellular signal transduction. In this context, analysis of the phosphoproteome is a prerequisite to better understand the cellular regulatory networks. Conventionally, due to the low contents of signaling proteins, selective enrichment of proteolytic phosphopeptides by immobilized metal affinity chromatography (IMAC) is performed prior to their LC-MS or -MS/MS analysis. Unfortunately, this technique still suffers from low selectivity and compromised analyte recoveries. To overcome these limitations, we propose IMAC systems comprising stationary phases based on collapsed Langmuir-Blodgett films of iron(III) stearate (FF) or iron(III) oxide nanoparticles (FO) and mobile phases relying on ammonia, piperidine and heptadecafluorooctanesulfonic acid (PFOS). Experiments with model phosphopeptides and phosphoprotein tryptic digests showed superior binding capacity, selectivity and recovery for both systems in comparison to the existing commercial analogs. As evidenced by LC-MS/MS analysis of the HeLa phosphoproteome, these features of the phases resulted in increased phosphoproteome coverage in comparison to the analogous commercially available phases, indicating that our IMAC protocol is a promising chromatographic tool for in-depth phosphoproteomic research. PMID:27016113

  10. Molecular insight in the purification of immunoglobulin by pseudobiospecific ligand l-histidine and histidyl moieties in histidine ligand affinity chromatography (HLAC) by molecular docking.

    PubMed

    Savane, Tushar S; Kumar, Sanjit; Janakiraman, Vignesh Narasimhan; Kamalanathan, Agamudi S; Vijayalakshmi, Mookambeswaran A

    2016-05-15

    Pseudobiospecific ligand l-histidine is an inexpensive, highly stable, non-toxic ligand explored successfully over the last twenty years for the purification of immunoglobulins in immobilised histidine ligand affinity chromatography. It is of great interest to know the molecular recognition sites of IgG to immobilized l-histidine. Here, we have used an in silico approach to explore the molecular recognition of l-histidine by IgG. We have assessed the feasible binding modes of histidine and its moieties at different sites of IgG and considered only those binding conformations which are exhibited via the imidazole ring NH group or any other OH donating group apart from the ones which are terminally conjugated with the support matrix. We categorised binding site into two categories; category I: inner binding groove and category II: surface binding groove and observed that the hinge region of IgG has most favourable binding pocket for l-histidine and histidyl moieties. Ser and Tyr residues on the hinge region make several significant interactions with l-histidine and histidyl moieties. In case of Fc region of IgG, l-histidine and histidyl moieties closely resemble the binding modes of Protein A, biomimetic ligand 22/8 and B domain of SpA to IgG. In addition to these we have also observed a significant binding site for l-histidine and histidyl moieties at Fab region of IgG. PMID:26476866

  11. Recognition and binding of β-lactam antibiotics to bovine serum albumin by frontal affinity chromatography in combination with spectroscopy and molecular docking.

    PubMed

    Li, Qian; Zhang, Tianlong; Bian, Liujiao

    2016-03-01

    Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical β-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV-vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22×10(1), 4.86×10(2) and 3.32×10(3) (L/mol), respectively. All the three β-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with β-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties. PMID:26882128

  12. Purification of α2-macroglobulin from Cohn Fraction IV by immobilized metal affinity chromatography: A promising method for the better utilization of plasma.

    PubMed

    Huangfu, Chaoji; Ma, Yuyuan; Lv, Maomin; Jia, Junting; Zhao, Xiong; Zhang, Jingang

    2016-07-01

    As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI-MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50°C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource. PMID:27214605

  13. Designed synthesis of Graphene @titania @mesoporous silica hybrid material as size-exclusive metal oxide affinity chromatography platform for selective enrichment of endogenous phosphopeptides.

    PubMed

    Yao, Jizong; Sun, Nianrong; Deng, Chunhui; Zhang, Xiangming

    2016-04-01

    In this work, a novel size-exclusive metal oxide affinity chromatography (SE-MOAC) platform was built for phosphoproteome research. The operation for preparing graphene @titania @mesoporous silica nanohybrids (denoted as G@TiO2@mSiO2) was facile and easy to conduct by grafting titania nanoparticles on polydopamine (PD)-covered graphene, following a layer of mesoporous silica was coated on the outermost layer. The G@TiO2@mSiO2 nanohybrids exhibited high sensitivity with a low detection limit of 5 amol/μL (a total amount of 1 fmol) and high selectivity for phosphopeptides at a mass ratio of phosphopeptides to non-phosphopeptides (1:1000). The size-exclusive capability of the nanohybrids were also demonstrated by enriching the phosphopeptides from the mixture of Bovine Serum Albumin (BSA), α-casein, and β-casein digests with a high mass ratio (β-casein digests: α-casein: BSA, 1:500:500), which was attributed to the large surface area and ordered mesoporous channels. In addition, the G@TiO2@mSiO2 nanohybrids were employed to capture the endogenous phosphopeptides from human serum successfully. PMID:26838411

  14. Development and characterization of the α3β4α5 nicotinic receptor cellular membrane affinity chromatography column and its application for on line screening of plant extracts.

    PubMed

    Ciesla, L; Okine, M; Rosenberg, A; Dossou, K S S; Toll, L; Wainer, I W; Moaddel, R

    2016-01-29

    The α3β4α5 nAChR has been recently shown to be a useful target for smoking cessation pharmacotherapies. Herein, we report on the development and characterization of the α3β4α5 nicotinic receptor column by frontal displacement chromatography. The binding affinity of the nicotine and minor alkaloids found in tobacco smoke condensates were determined for both the α3β4 and α3β4α5 nicotinic receptors. It was demonstrated that while no subtype selectivity was observed for nicotine and nornicotine, anabasine was selective for the α3β4α5 nicotinic receptor. The non-competitive inhibitor binding site was also studied and it was demonstrated while mecamylamine was not selective between subtypes, buproprion showed subtype selectivity for the α3β4 nicotinic receptor. The application of this methodology to complex mixtures was then carried out by screening aqueous-alcoholic solutions of targeted plant extracts, including Lycopodium clavatum L. (Lycopodiaceae) and Trigonella foenum graecum L. (Fabaceae) against both the α3β4 and α3β4α5 nAChRs. PMID:26774122

  15. Target-directed screening of the bioactive compounds specifically binding to β₂-adrenoceptor in Semen brassicae by high-performance affinity chromatography.

    PubMed

    An, Yuxin; Li, Xia; Sun, Huanmei; Bian, Wenhai; Li, Zijian; Zhang, Youyi; Zhao, Xinfeng; Zheng, Xiaohui

    2015-10-01

    The bioactive ingredients in Semen sinapis were rapidly screened by immobilized β2-adrenoceptor (β2-AR) and target-directed molecular docking. The methods involved the attachment of β2-AR using any amino group in the receptor, the simultaneous separation and identification of the retention compounds by high-performance affinity chromatography; the binding mechanism of the interesting compound to the receptor was investigated by zonal elution and molecular docking. Sinapine in Semen sinapis was proved to be the bioactive compound that specifically binds to the immobilized receptor. The association constant of sinapine to β2-AR was determined to be 1.36 × 10(5)  M(-1) with a value of 1.27 × 10(-6)  M for the number of binding sites. Ionic bond was believed to be the driving force during the interaction between sinapine and β2-AR. It is possible to become a powerful alternative for rapid screening of bioactive compounds from a complex matrix such as traditional Chinese medicine and further investigation on the drug-receptor interaction. PMID:25982051

  16. Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481

    PubMed Central

    2012-01-01

    The aminopeptidase P (PepP, EC 3.4.11.9) gene from Lactococcus lactis ssp. lactis DSM 20481 was cloned, sequenced and expressed recombinantly in E. coli BL21 (DE3) for the first time. PepP is involved in the hydrolysis of proline-rich proteins and, thus, is important for the debittering of protein hydrolysates. For accurate determination of PepP activity, a novel gas chromatographic assay was established. The release of L-leucine during the hydrolysis of L-leucine-L-proline-L-proline (LPP) was examined for determination of PepP activity. Sufficient recombinant PepP production was achieved via bioreactor cultivation at 16 °C, resulting in PepP activity of 90 μkatLPP Lculture-1. After automated chromatographic purification by His-tag affinity chromatography followed by desalting, PepP activity of 73.8 μkatLPP Lculture-1 was achieved. This was approximately 700-fold higher compared to the purified native PepP produced by Lactococcus lactis ssp. lactis NCDO 763 as described in literature. The molecular weight of PepP was estimated to be ~ 40 kDa via native-PAGE together with a newly developed activity staining method and by SDS-PAGE. Furthermore, the kinetic parameters Km and Vmax were determined for PepP using three different tripeptide substrates. The purified enzyme showed a pH optimum between 7.0 and 7.5, was most active between 50°C and 60°C and exhibited reasonable stability at 0°C, 20°C and 37°C over 15 days. PepP activity could be increased 6-fold using 8.92 mM MnCl2 and was inhibited by 1,10-phenanthroline and EDTA. PMID:22853547

  17. Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

    PubMed Central

    Bieberich, Erhard

    2011-01-01

    The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane

  18. 32P-postlabeling detection of thymine glycols: evaluation of adduct recoveries after enhancement with affinity chromatography, nuclease P1, nuclease S1, and polynucleotide kinase.

    PubMed

    Reddy, M V; Bleicher, W T; Blackburn, G R

    1991-04-01

    Thymine glycol (Tg) is a product of DNA damage by oxygen radicals generated by oxidative mutagens and carcinogens and ionizing radiation. The highly sensitive 32P-postlabeling assay was validated and optimized for the measurement of Tg generated in vitro by the reaction of dTp or calf thymus DNA with osmium tetroxide (OsO4). Adduct detection was enhanced by purification of Tg adducts using phenylboronate affinity chromatography or by preferential dephosphorylation of unmodified 3'-nucleotides with nuclease P1, nuclease S1, or polynucleotide kinase; Tg nucleotides were found to be resistant to limited enzymatic 3'-dephosphorylation. Two adducts were seen with OsO4-modified dTp, which may have been cis-Tg adducts, because they were retained on a phenylboronate column, and because OsO4 selectively forms cis-Tg adducts. With OsO4-modified DNA, several adducts were detected, two major derivatives of which coincided chromatographically with those seen in OsO4-modified dTp. The recoveries of major adducts were similar before and after enrichment by different methods, indicating that Tg adducts were resistant to enzymatic dephosphorylation. The efficacy of labeling of the two major Tg adducts by polynucleotide kinase was optimal at 60 microM ATP and higher, whereas it was about 3%, 50%, and 80% of the optimal rate at 2, 10, and 30 microM, respectively. This was in contrast to our previous finding that only 0.25 microM ATP was needed for optimal labeling of benzoquinone-DNA adducts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2025496

  19. Characterization of aquatic humic substances and their metal complexes by immobilized metal-chelate affinity chromatography on iron(III)-loaded ion exchangers.

    PubMed

    Burba, P; Jakubowski, B; Kuckuk, R; Küllmer, K; Heumann, K G

    2000-12-01

    The analytical fractionation of aquatic humic substances (HS) by means of immobilized metal-chelate affinity chromatography (IMAC) on metal-loaded chelating ion exchangers is described. The cellulose HYPHAN, loaded with different trivalent ions, and the chelate exchanger Chelex 100, loaded to 90% of its capacity with Fe(III), were used. The cellulose HYPHAN, loaded with 2% Fe(III), resulted in HS distribution coefficients Kd of up to 10(3.7) mL/g at pH 4.0 continuously decreasing down to 10(1.5) at pH 12, which were appropriate for HS fractionation by a pH-depending chromatographic procedure. Similar distribution coefficients Kd were obtained for HS sorption onto Fe(III)-loaded Chelex 100. On the basis of Fe-loaded HYPHAN both, a low-pressure and high-pressure IMAC technique, were developed for the fractionation of dissolved HS applying a buffer-based pH gradient for their gradual elution between pH 4.0 and 12.0. By coupling the Chelex 100 column under high-pressure conditions with an inductively coupled plasma mass spectrometer an on-line characterization of HS metal species could be achieved. Using these fractionation procedures a number of reference HS were characterized. Accordingly, the HA (humic acids) and FA (fulvic acids) studied could be discriminated into up to 6 fractions by applying cellulose HYPHAN, significantly differing in their Cu(II) complexation capacity but hardly in their substructures assessed by conventional FTIR. In the case of using Chelex 100 exchanger resin two major UV active HS fractions were obtained, which significantly differ in their complexation properties for Cu(II) and Pb(II), respectively. PMID:11227549

  20. [Determination of the interaction kinetics between meloxicam and β-cyclodextrin using the quantitative high-performance affinity chromatography coupled with mass spectrometry].

    PubMed

    Wang, Cai-fen; Li, Zhuo; Wang, Xiao-bo; Li, Hai-yan; Zhang, Ji-wen; Sun, Li-xin

    2015-09-01

    The association rate constant and dissociation rate constant are important parameters of the drug-cyclodextrin supermolecule systems, which determine the dissociation of drugs from the complex and the further in vivo absorption of drugs. However, the current studies of drug-cyclodextrin interactions mostly focus on the thermodynamic parameter of equilibrium constants (K). In this paper, a method based on quantitative high performance affinity chromatography coupled with mass spectrometry was developed to determine the apparent dissociation rate constant (k(off,app)) of drug-cyclodextrin supermolecule systems. This method was employed to measure the k(off,app) of meloxicam and acetaminophen. Firstly, chromatographic peaks of drugs and non-retained solute (uracil) on β-cyclodextrin column at different flow rates were acquired, and the retention time and variance values were obtained via the fitting the peaks. Then, the plate heights of drugs (H(R)) and uracil (H(M,C)) were calculated. The plate height of theoretical non-retained solute (H(M,T)) was calculated based on the differences of diffusion coefficient and the stagnant mobile phase mass transfer between drugs and uracil. Finally, the k(off,app) was calculated from the slope of the regression equation between (H(R)-H(M,T)) and uk/(1+k)2, (0.13 ± 0.00) s(-1) and (4.83 ± 0.10) s(-1) for meloxicam and acetaminophen (control drug), respectively. In addition, the apparent association rate constant (k(on,app)) was also calculated through the product of K (12.53 L x mol(-1)) and k(off,app). In summary, it has been proved that the method established in our study was simple, efficiently fast and reproducible for investigation on the kinetics of drug-cyclodextrin interactions. PMID:26757555

  1. Determination of a highly selective mixed-affinity sigma receptor ligand, in rat plasma by ultra performance liquid chromatography mass spectrometry and its application to a pharmacokinetic study

    PubMed Central

    Jamalapuram, Seshulatha; Vuppala, Pradeep K.; Mesangeau, Christophe; McCurdy, Christopher R.; Avery, Bonnie A.

    2014-01-01

    A selective, rapid and sensitive ultra performance liquid chromatography mass spectrometry (UPLC/MS) method was developed and validated to quantitate a highly selective mixed-affinity sigma receptor ligand, CM156 (3-(4-(4-cyclohexylpiperazin-1-yl)butyl)benzo[d] thiazole-2(3H)-thione), in rat plasma. CM156 and the internal standard (aripiprazole) were extracted from plasma samples by a single step liquid–liquid extraction using chloroform. The analysis was carried out on an ACQUITY UPLCTM BEH HILIC column (1.7 µm, 2.1 mm × 50 mm) with isocratic elution at flow rate of 0.2 mL/min using 10 mM ammonium formate in 0.1% formic acid and acetonitrile (10:90) as the mobile phase. The detection of the analyte was performed on a mass spectrometer operated in selected ion recording (SIR) mode with positive electrospray ionization (ESI). The validated analytical method resulted in a run time of 4 min and the retention times observed were 2.6 ± 0.1 and 2.1 ± 0.1 min for CM156 and the IS, respectively. The calibration curve exhibited excellent linearity over a concentration range of 5–4000 ng/mL with the lower limit of quantification of 5 ng/mL. The intra- and inter-day precision values were below 15% and accuracy ranged from −6.5% to 5.0%. The mean recovery of CM156 from plasma was 96.8%. The validated method was applied to a pilot intravenous pharmacokinetic study in rats. PMID:22406103

  2. sup 32 P-postlabeling detection of thymine glycols: evaluation of adduct recoveries after enhancement with affinity chromatography, nuclease P1, nuclease S1, and polynucleotide kinase

    SciTech Connect

    Reddy, M.V.; Bleicher, W.T.; Blackburn, G.R. )

    1991-04-01

    Thymine glycol (Tg) is a product of DNA damage by oxygen radicals generated by oxidative mutagens and carcinogens and ionizing radiation. The highly sensitive {sup 32}P-postlabeling assay was validated and optimized for the measurement of Tg generated in vitro by the reaction of dTp or calf thymus DNA with osmium tetroxide (OsO{sub 4}). Adduct detection was enhanced by purification of Tg adducts using phenylboronate affinity chromatography or by preferential dephosphorylation of unmodified 3'-nucleotides with nuclease P1, nuclease S1, or polynucleotide kinase; Tg nucleotides were found to be resistant to limited enzymatic 3'-dephosphorylation. Two adducts were seen with OsO{sub 4}-modified dTp, which may have been cis-Tg adducts, because they were retained on a phenylboronate column, and because OsO{sub 4} selectively forms cis-Tg adducts. With OsO{sub 4}-modified DNA, several adducts were detected, two major derivatives of which coincided chromatographically with those seen in OsO{sub 4}-modified dTp. The recoveries of major adducts were similar before and after enrichment by different methods, indicating that Tg adducts were resistant to enzymatic dephosphorylation. The efficacy of labeling of the two major Tg adducts by polynucleotide kinase was optimal at 60 microM ATP and higher, whereas it was about 3%, 50%, and 80% of the optimal rate at 2, 10, and 30 microM, respectively. This was in contrast to our previous finding that only 0.25 microM ATP was needed for optimal labeling of benzoquinone-DNA adducts.

  3. Simultaneous high-throughput determination of interaction kinetics for drugs and cyclodextrins by high performance affinity chromatography with mass spectrometry detection.

    PubMed

    Wang, Caifen; Wang, Xiaobo; Xu, Xiaonan; Liu, Botao; Xu, Xu; Sun, Lixin; Li, Haiyan; Zhang, Jiwen

    2016-02-25

    The individual determination of the apparent dissociation rate constant (kd,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate tests and massive data fitting, unable to provide the apparent association rate constant (ka) and equilibrium binding constant (Ka). In this study, a HPAC with mass spectrometry detection (HPAC-MS/MS) was employed to determine the drug-cyclodextrin (CD) interaction kinetics with low sample loading quantity (<10 ng per injection for single compound) and high-throughput yield as twenty drugs determined in one injection. The kd,app measured by HPAC-MS/MS approach were 0.89 ± 0.07, 4.34 ± 0.01, 1.48 ± 0.01 and 7.77 ± 0.04 s(-1) for ketoprofen, trimethoprim, indapamide and acetaminophen, with kd,app for acetaminophen consistent with that from the HPAC method with UV detector in our previous studies. For twenty drugs with diverse structures and chemical properties, good correlationship was found between kd,app measured by single compound analysis method and high-throughput HPAC-MS/MS approach, with the correlation coefficient of 0.987 and the significance F less than 0.001. Comprehensive quantification of ka,app, kd,app and Ka values was further performed based on the measurement of kd,app by peak profiling method and Ka by the peak fitting method. And the investigation of the drug-CD interaction kinetics under different conditions indicated that the column temperature and mobile phase composition significantly affected the determination of ka,app, kd,app and Ka while also dependent on the acidity and basicity of drugs. In summary, the high-throughput HPAC-MS/MS approach has been demonstrated high efficiency in determination of the drug-CD primary interaction kinetic parameter, especially, kd,app, being proven as a novel tool in screening the right CD for the solubilization of the right drug. PMID:26851087

  4. Purification of adenovirus hexon by high performance liquid chromatography.

    PubMed

    Siegel, S A; Hutchins, J E; Witt, D J

    1987-09-01

    Hexon is the major structural protein of adenovirus, and has significance in studies of virus structure and function, vaccine development, and immunodiagnosis. We describe a simple, single-step, anion-exchange high performance liquid chromatography (HPLC) method for the high yield purification of hexon. Purity of the isolated hexon was assessed by SDS-PAGE and HPLC methods. The isolated hexon was immunologically reactive with anti-hexon monoclonal antibody in a dot-blot assay. It also retained immunogenicity, as polyclonal antisera from rabbits immunized with hexon showed the desired antigen specificity. The enhanced speed of this purification method allows for the efficient isolation of hexon from various serotypes, and thus may facilitate comparative studies of hexon immunobiology. PMID:3680460

  5. Open tubular columns containing the immobilized ligand binding domain of peroxisome proliferator-activated receptors α and γ for dual agonists characterization by frontal affinity chromatography with MS detection

    PubMed Central

    Temporini, C.; Pochetti, G.; Fracchiolla, G.; Piemontese, L.; Montanari, R.; Moaddel, R.; Laghezza, A.; Altieri, F.; Cervoni, L.; Ubiali, D.; Prada, E.; Loiodice, F.; Massolini, G.; Calleri, E.

    2013-01-01

    The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. In the last years novel PPARs ligands have been identified and these include PPARα/γ dual agonists. To rapidly identify novel PPARs dual ligands, a robust binding assay amenable to high-throughput screening towards PPAR isoforms would be desirable. In this work we describe a parallel assay based on the principles of Frontal Affinity Chromatography coupled to Mass Spectrometry (FAC-MS) that can be used to characterize dual agonists. For this purpose the ligand binding domain of PPARα receptor was immobilized onto the surface of open tubular capillaries to create new PPAR-alpha-OT columns to be used in parallel with PPAR-gamma-OT columns. The two biochromatographic systems were used in both ranking and Kd experiments towards new ureidofibrate-like dual agonists for subtype selectivity ratio determination. In order to validate the system, the Kd values determined by frontal analysis chromatography were compared to the affinity constants obtained by ITC experiments. The results of this study strongly demonstrate the specific nature of the interaction of the ligands with the two immobilized receptor subtypes. PMID:23466198

  6. FYWHCLDE-based affinity chromatography of IgG: effect of ligand density and purifications of human IgG and monoclonal antibody.

    PubMed

    Zhao, Wei-Wei; Shi, Qing-Hong; Sun, Yan

    2014-08-15

    This work reports the development of an octapeptide-based affinity adsorbent for the purification of human IgG (hIgG) and monoclonal antibody (mAb). The octapeptide was FYWHCLDE selected earlier by the biomimetic design of affinity peptide ligands for hIgG. The ligand was coupled to Sepharose gel at four densities from 10.4 to 31.0μmol/mL, and the effect of peptide density on the adsorption of hIgG and bovine serum albumin (BSA) was first investigated. The binding capacity of hIgG increased from 104.2 to 176.4mg/mL within the ligand density range, and the binding affinity (dissociation constant) kept at 2.4-3.7μM. Batch adsorption revealed that the selectivity of FYWHCLDE-Sepharose for IgG was 30-40 times over BSA. The effective pore diffusivity of IgG decreased somewhat with increasing ligand density, but the dynamic binding capacity at 10% breakthrough, measured by using 10-fold diluted human serum as feedstock, doubled with increasing ligand density from 10.4 to 31.0μmol/mL due to the remarkable increase of static binding capacity. By using the affinity column with a ligand density of 23.9μmol/mL, hIgG and humanized mAb purifications from human serum and cell culture supernatant, respectively, were achieved at high purities and recovery yields. Finally, the robustness of the peptide gel was demonstrated by recycled use of the affinity column in 20 breakthrough cycles. PMID:24947889

  7. [Determination of peptide and protein diversity in venom of the spider Selenocosmia jiafu by high performance liquid chromatography and mass spectrometry].

    PubMed

    Hu, Zhaotun; Xiao, Zhen; Zhou, Xi; Chen, Jia; Chen, Bo; Liu, Zhonghua

    2015-06-01

    Selenocosmia jiafu (S. jiafu) is recently identified as a new species of spider in P. R. China. These medium bodied venomous spiders are distributed mainly in the hilly areas of southwest of China, mostly at Yunnan and Guangxi Provinces. In order to understand the composition of the S. jiafu venom, we performed a preliminary analysis of this venom using reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The S. jiafu venom was separated by RP-HPLC in an analytical C18 column (phenomenex 100 A, 250 mm x 4.6 mm, 5 µm) equilibrated with solution A (distilled water with 0.1% trifluoroacetic acid), using a gradient from 0% to 50% of solution B (acetonitrile with 0.1% trifluoroacetic acid) over 50 min with a flow rate of 1 mL/min. The isolated venom proteins were treated with in-gel digestion separated by SDS- PAGE and then identified by liquid chromatography-electrospray ionization quadrupole-time of flight mass spectrometry (LC-ESI-QTOF-MS) techniques. The results show that more than 40 fractions eluted were monitored at 215 nm in the RP-HPLC chromatogram of the venom of the spider S. jiafu. Most of the fractions were eluted with retention times of 5-15 min and 25-40 min, corresponding to 5%-15% and 25%-40% acetonitrile, respectively. The venom contains 238 peptides that follow a bimodal distribution, with about 62.5% of the peptides having a relative molecular mass of 3,000-4,500 and about 33.2% of the peptides having a relative molecular mass of 1,000-3,000. This distribution model is rather different from those of peptides from other tarantula spider venoms analyzed. To explore the relative molecular mass distribution of the venom proteins, the venom was analyzed by SDS-PAGE using standard protocols. Except for peptides with relative molecular mass lower than 10,000, the SDS-PAGE

  8. The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) insect cells

    PubMed Central

    Bhatia, Prateek A.; Moaddel, Ruin; Wainer, Irving W.

    2010-01-01

    CMAC (cellular membrane affinity chromatography columns) have been developed for the study of the human multidrug transporters MRP1, MRP2 and the breast cancer resistance protein (BCRP). The columns were constructed using the immobilized artificial membrane (IAM) stationary phase and cellular membrane fragments obtained from Spodopetra frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp c-DNA, using a baculovirus expression system. The resulting CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns and a control column produced using membrane fragments from non-transfected Sf9 cells, CMAC(Sf9), were characterized using frontal affinity chromatography using [3H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9MRP1) column, etoposide and furosemide on the CMAC(Sf9MRP2) column and etoposide and fumitremorgin C on the CMAC(Sf9BCPR) column The binding affinities (Ki values) obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. (S)-verapamil displaced [3H]-etoposide on the CMAC(Sf9MRP1) column to a greater extent than (R)-verapamil and the relative IC50 values of the enantiomers were calculated using the changes in the retention times of the marker. The observed enantioselectivity and calculated IC50 values were consistent with previously reported data. The results indicated that the CMAC(Sf9MRP1), CMAC(Sf9MRP2) and CMAC(Sf9BCRP) columns can be used for the study of binding to the MRP1, MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This is the first example of the use of membranes from a non-mammalian cell line in an affinity chromatographic system. PMID:20441926

  9. Short communication: Identification of iron-binding peptides from whey protein hydrolysates using iron (III)-immobilized metal ion affinity chromatography and reversed phase-HPLC-tandem mass spectrometry.

    PubMed

    Cruz-Huerta, Elvia; Martínez Maqueda, Daniel; de la Hoz, Lucia; da Silva, Vera S Nunes; Pacheco, Maria Teresa Bertoldo; Amigo, Lourdes; Recio, Isidra

    2016-01-01

    Peptides with iron-binding capacity obtained by hydrolysis of whey protein with Alcalase (Novozymes, Araucaria, PR, Brazil), pancreatin, and Flavourzyme (Novozymes) were identified. Hydrolysates were subjected to iron (III)-immobilized metal ion affinity chromatography, and the bound peptides were sequenced by mass spectrometry. Regardless of the enzyme used, the domains f(42-59) and f(125-137) from β-lactoglobulin enclosed most of identified peptides. This trend was less pronounced in the case of peptides derived from α-lactalbumin, with sequences deriving from diverse regions. Iron-bound peptides exhibited common structural characteristics, such as an abundance of Asp, Glu, and Pro, as revealed by mass spectrometry and AA analysis. In conclusion, this characterization of iron-binding peptides helps clarify the relationship between peptide structure and iron-chelating activity and supports the promising role of whey protein hydrolysates as functional ingredients in iron supplementation treatments. PMID:26601589

  10. Phenotyping breast cancer cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis and affinity chromatography for glutathione-binding proteins

    PubMed Central

    2010-01-01

    Background Transformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs) and other proteins interacting with glutathione (GSH) in model cell lines could be of particular interest. Methods We compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE). We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots. Results Correlation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics. Conclusions Our results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines. PMID:20731849

  11. Characteristics of an Extensive Mycobacterium avium subspecies paratuberculosis Recombinant Protein Set

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the first step of a comprehensive large-scale antigen discovery project, 651 Mycobacterium avium subspecies paratuberculosis proteins were produced in Escherichia coli. All of these were purified by affinity chromatography, dialyzed in phosphate buffered saline, and analyzed on SDS-PAGE gels. C...

  12. Combined docking, molecular dynamics simulations and spectroscopic studies for the rational design of a dipeptide ligand for affinity chromatography separation of human serum albumin.

    PubMed

    Aghaee, Elham; Ghasemi, Jahan B; Manouchehri, Firouzeh; Balalaie, Saeed

    2014-10-01

    A computational approach to designing a peptide-based ligand for the purification of human serum albumin (HSA) was undertaken using molecular docking and molecular dynamics (MD) simulation. A three-step procedure was performed to design a specific ligand for HSA. Based on the candidate pocket structure of HSA (warfarin binding site), a peptide library was built. These peptides were then docked into the pocket of HSA using the GOLD program. The GOLDscore values were used to determine the affinity of peptides for HSA. Consequently, the dipeptide Trp-Trp, which shows a high GOLDscore value, was selected and linked to a spacer arm of Lys[CO(CH2)5NH] on the surface of ECH-lysine sepharose 4 gel. For further evaluation, the Autodock Vina program was used to dock the linked compound into the pocket of HSA. The docking simulation was performed to obtain a first guess of the binding structure of the spacer-Trp-Trp-HSA complex and subsequently analyzed by MD simulations to assess the reliability of the docking results. These MD simulations indicated that the ligand-HSA complex remains stable, and water molecules can bridge between the ligand and the protein by hydrogen bonds. Finally, absorption spectroscopic studies were performed to illustrate the appropriateness of the binding affinity of the designed ligand toward HSA. These studies demonstrate that the designed dipeptide can bind preferentially to the warfarin binding site. PMID:25220335

  13. Leukotriene-E4 in human urine: Comparison of on-line purification and liquid chromatography-tandem mass spectrometry to affinity purification followed by enzyme immunoassay.

    PubMed Central

    Armstrong, Michael; Liu, Andrew H.; Harbeck, Ronald; Reisdorph, Rick; Rabinovitch, Nathan; Reisdorph, Nichole

    2009-01-01

    A new analytical method suitable for high throughput measurements of LTE4 in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/ tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500 pg/mL in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE4 from healthy adults and children are reported. PMID:19726242

  14. Preparative separation of 1,3,6-pyrenetrisulfonic acid trisodium salt from the color additive D&C Green No. 8 by affinity-ligand pH-zone-refining counter-current chromatography

    PubMed Central

    Weisz, Adrian; Mazzola, Eugene P.; Ito, Yoichiro

    2011-01-01

    In developing analytical methods for batch certification of the color additive D&C Green No. 8 (G8), the U.S. Food and Drug Administration needed the trisodium salt of 1,3,6-pyrenetrisulfonic acid (P3S) for use as a reference material. Since P3S was not commercially available, preparative quantities of it were separated from portions of a sample of G8 that contained ~ 3.5% P3S. The separations were performed by affinity-ligand pH-zone-refining counter-current chromatography using dodecylamine (DA) as the ligand. The added ligand enabled partitioning of the polysulfonated components into the organic stationary phase of the two-phase solvent system used, 1-butanol – water (1:1). A typical separation that involved 20.3 g of G8, using sulfuric acid as the retainer acid and 20% DA in the stationary phase and 0.1M sodium hydroxide as the mobile phase, resulted in ~0.58 g of P3S of greater than 99% purity. The identification and characterization of the separated P3S were performed by proton nuclear magnetic resonance, high-resolution mass spectrometry, ultra-violet spectra and high-performance liquid chromatography. PMID:21982993

  15. Identification of homologous pairing and strand-exchange activity from a human tumor cell line based on Z-DNA affinity chromatography

    SciTech Connect

    Fishel, R.A.; Detmer, K.; Rich, A.

    1988-01-01

    An enzymatic activity that catalyzes ATP-dependent homologous pairing and strand exchange of duplex linear DNA and single-stranded circular DNA has been purified several thousand-fold from a human leukemic T-lymphoblast cell line. The activity was identified after chromatography of nuclear proteins on a Z-DNA column matrix. The reaction was shown to transfer the complementary single strand from a donor duplex linear substrate to a viral circular single-stranded acceptor beginning at the 5' end and proceeding in the 3' direction. Products of the strand-transfer reaction were characterized by electron microscopy. A 74-kDa protein was identified as the major ATP-binding peptide in active strand transferase fractions. The protein preparation described in this report binds more strongly to Z-DNA than to B-DNA.

  16. Affinity capture of [Arg8]vasopressin-receptor complex using immobilized antisense peptide.

    PubMed Central

    Lu, F X; Aiyar, N; Chaiken, I

    1991-01-01

    Solubilized noncovalent complexes of [Arg8]-vasopressin (AVP) with receptor proteins from rat liver membranes were isolated by selective binding to silica-immobilized antisense (AS) peptide. The affinity chromatographic support was prepared with a chemically synthesized AS peptide whose sequence is encoded by the AS DNA corresponding to the 20 amino-terminal residues of the AVP bovine neurophysin II biosynthetic precursor [pro-AVP/BNPII-(20-1)], a region that includes the AVP sequence at residues 1-9. The AVP-related AS peptide previously was shown to bind selectively to AVP. The AS peptide-AVP interaction mechanism hypothesized, contact by hydropathic complementarily at multiple sites along the peptide chains, led to the prediction that AVP bound to its receptor would still have enough free surface to interact with immobilized AS peptide. To test this prediction of a three-way interaction, [3H]AVP-receptor was obtained as a solubilized, partially purified fraction from rat liver membrane. When this fraction was eluted through AS pro-AVP/BNPII-(20-1) silica, a complex containing [3H]AVP was bound and separated from the major, unretarded membrane protein fraction as well as from free AVP. Chemical crosslinking of [3H]AVP complex, SDS/PAGE of the products, and analysis of gel slices by scintillation counting led to detection of two major radiolabeled bands of 31 and 38 kDa. Covalent labeling was blocked when unlabeled AVP was added as a competitor before crosslinking. A third radiolabeled protein band of 15 kDa was found when the receptor complex was solubilized from rat liver membranes in the absence of the protease inhibitor phenylmethylsulfonyl fluoride. Covalently crosslinked [3H]AVP complex also was bound to the AS peptide column; binding was blocked by competition with unlabeled AVP in the elution buffer. Since the AVP-linked 31- and 38-kDa proteins have the same apparent molecular mass on SDS/PAGE as found previously by photo-affinity labeling, we conclude

  17. Dataset of gene cloning and gel filtration chromatography of R-est6.

    PubMed

    Soni, Surabhi; Odaneth, Annamma A; Lali, Arvind M; Chandrayan, Sanjeev K

    2016-06-01

    The data presented in this article are connected to the research article entitled "Expression, purification and biochemical characterization of a family 6 carboxylesterase from Methylococcus capsulatus (bath)" (Soni et al., 2016 [1]). The family 6 carboxylesterases are the smallest and display broad substrate specificity. The 1 kb gene encoding, a family 6 carboxylesterase - R-est6, was amplified from the genome of M. capsulatus (bath strain), and showed in the agarose gel. The corresponding purified protein, after overexpression in Escherichia coli, was biochemically studied in the research article (Soni et al., 2016 [1]). R-est6 has hydrophobic patches on the surface so, it is expected to show oligimeric forms. Here, we have confirmed the presence of oligomers by gel filtration chromatography data and the proteins belonging to the different peaks are shown on a SDS-PAGE. PMID:27222859

  18. Dataset of gene cloning and gel filtration chromatography of R-est6

    PubMed Central

    Soni, Surabhi; Odaneth, Annamma A.; Lali, Arvind M.; Chandrayan, Sanjeev K.

    2016-01-01

    The data presented in this article are connected to the research article entitled “Expression, purification and biochemical characterization of a family 6 carboxylesterase from Methylococcus capsulatus (bath)” (Soni et al., 2016 [1]). The family 6 carboxylesterases are the smallest and display broad substrate specificity. The 1 kb gene encoding, a family 6 carboxylesterase – R-est6, was amplified from the genome of M. capsulatus (bath strain), and showed in the agarose gel. The corresponding purified protein, after overexpression in Escherichia coli, was biochemically studied in the research article (Soni et al., 2016 [1]). R-est6 has hydrophobic patches on the surface so, it is expected to show oligimeric forms. Here, we have confirmed the presence of oligomers by gel filtration chromatography data and the proteins belonging to the different peaks are shown on a SDS-PAGE. PMID:27222859

  19. Functional Characterization of the Kinase Activation Loop in Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Using Tandem Affinity Purification and Liquid Chromatography-Mass Spectrometry*

    PubMed Central

    Wang, Peng; Wu, Fang; Ma, Yupo; Li, Liang; Lai, Raymond; Young, Leah C.

    2010-01-01

    Previous studies have shown that the kinase activation loop (KAL) of the oncogenic fusion protein NPM-ALK regulates its overall tyrosine phosphorylation status and tumorigenicity. Using tandem affinity purification-mass spectrometry, we assessed how the KAL of NPM-ALK regulates the phosphorylation status of its individual tyrosines. Using the lysates of GP293 cells transfected with NPM-ALK, our highly reproducible results showed evidence of phosphorylation in all 3 tyrosines in KAL and 8 tyrosines outside KAL. We created 7 KAL mutants, each of which carried a Tyr-to-Phe mutation of ≥1 of the 3 tyrosines in KAL. A complete loss of the 8 phosphotyrosines outside KAL was found in 3 KAL mutants, and their oncogenicity (assessed by cell viability, colony formation, and the ability to phosphorylate effector proteins) was abrogated. A partial loss of the 8 phosphotyrosines was found in 4 KAL mutants, but their oncogenicity did not show simple correlation with the number of residual phosphotyrosines. Tyr-to-Phe mutations of each of the 8 phosphotyrosines outside KAL did not result in a significant decrease in the oncogenicity. In conclusion, we have provided details of how the KAL in NPM-ALK regulates its tyrosine phosphorylation pattern. Our results challenge some of the current concepts regarding the relationship between the tyrosine phosphorylation and oncogenicity of NPM-ALK. PMID:19887368

  20. CHARACTERIZATION OF INTERACTION KINETICS BETWEEN CHIRAL SOLUTES AND HUMAN SERUM ALBUMIN BY USING HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PEAK PROFILING

    PubMed Central

    Tong, Zenghan; Hage, David S.

    2011-01-01

    Peak profiling and high-performance columns containing immobilized human serum albumin (HSA) were used to study the interaction kinetics of chiral solutes with this protein. This approach was tested using the phenytoin metabolites 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) as model analytes. HSA columns provided some resolution of the enantiomers for each phenytoin metabolite, which made it possible to simultaneously conduct kinetic studies on each chiral form. The dissociation rate constants for these interactions were determined by using both the single flow rate and multiple flow rate peak profiling methods. Corrections for non-specific interactions with the support were also considered. The final estimates obtained at pH 7.4 and 37°C for the dissociation rate constants of these interactions were 8.2–9.6 s−1 for the two enantiomers of m-HPPH and 3.2–4.1 s−1 for the enantiomers of p-HPPH. These rate constants agreed with previous values that have been reported for other drugs and solutes that have similar affinities and binding regions on HSA. The approach used in this report was not limited to phenytoin metabolites or HSA but could be applied to a variety of other chiral solutes and proteins. This method could also be adopted for use in the rapid screening of drug-protein interactions. PMID:21872871

  1. On-line coupling of surface plasmon resonance optical sensing to size-exclusion chromatography for affinity assessment of antibody samples.

    PubMed

    Lakayan, Dina; Haselberg, Rob; Niessen, Wilfried M A; Somsen, Govert W; Kool, Jeroen

    2016-06-24

    Surface plasmon resonance (SPR) is an optical technique that measures biomolecular interactions. Stand-alone SPR cannot distinguish different binding components present in one sample. Moreover, sample matrix components may show non-specific binding to the sensor surface, leading to detection interferences. This study describes the development of coupled size-exclusion chromatography (SEC) SPR sensing for the separation of sample components prior to their on-line bio-interaction analysis. A heterogeneous polyclonal human serum albumin antibody (anti-HSA) sample, which was characterized by proteomics analysis, was used as test sample. The proposed SEC-SPR coupling was optimized by studying system parameters, such as injection volume, flow rate and sample concentration, using immobilized HSA on the sensor chip. Automated switch valves were used for on-line regeneration of the SPR sensor chip in between injections and for potential chromatographic heart cutting experiments, allowing SPR detection of individual components. The performance of the SEC-SPR system was evaluated by the analysis of papain-digested anti-HSA sampled at different incubation time points. The new on-line SEC-SPR methodology allows specific label-free analysis of real-time interactions of eluting antibody sample constituents towards their antigenic target. PMID:27215465

  2. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes. PMID:25764651

  3. Use of affinity-directed liquid chromatography-mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction

    PubMed Central

    Griffiths, Amy E.; Wang, Wensheng; Hagen, Fred K.; Fay, Philip J.

    2011-01-01

    Summary Background Neutralizing factor (F) VIII antibodies develop in ~30% of individuals with hemophilia A and show specificity to multiple sites in the FVIII protein. Methods Reactive epitopes to an immobilized IgG fraction prepared from a high-titer, FVIII inhibitor plasma were determined following immuno-precipitation (IP) of tryptic and chymotryptic peptides derived from digests of the A1 and A2 subunits of FVIIIa and FVIII light chain. Peptides were detected and identified using highly sensitive liquid chromatography-mass spectrometry (LC-MS). Results Coverage maps of the A1 subunit, A2 subunit and light chain represented 79%, 69% and 90%, respectively, of the protein sequences. Dot blots indicated that the inhibitor IgG reacted with epitopes contained within each subunit of FVIIIa. IP coupled with LC-MS identified 19 peptides representing epitopes from all FVIII A and C domains. The majority of peptides (10) were derived from the A2 domain. Three peptides mapped to the C2 domain, while two mapped to the A1 and A3 domains, and single peptides mapped to the a1 segment and C1 domain. Epitopes were typically defined by peptide sequences of <12 residues. Conclusions IP coupled with LC-MS identified extensive antibody reactivity at high resolution over the entire functional FVIII molecule and yielded sequence lengths of less than 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein-protein and protein-membrane interactions. PMID:21668738

  4. Semi-quantitative Measurement of a Specific Glycoform Using a DNA-tagged Antibody and Lectin Affinity Chromatography for Glyco-biomarker Development*

    PubMed Central

    Lee, Ju Hee; Cho, Chang Hee; Kim, Sun Hee; Kang, Jeong Gu; Yoo, Jong Shin; Chang, Chulhun Ludgerus; Ko, Jeong-Heon; Kim, Yong-Sam

    2015-01-01

    Aberrant glycosylation-targeted disease biomarker development is based on cumulative evidence that certain glycoforms are mass-produced in a disease-specific manner. However, the development process has been hampered by the absence of an efficient validation method based on a sensitive and multiplexed platform. In particular, ELISA-based analytical tools are not adequate for this purpose, mainly because of the presence of a pair of N-glycans of IgG-type antibodies. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. This analytical method was applied to an in vitro diagnostic multivariate index assay where a panel of hepatocellular carcinoma (HCC) biomarkers comprising alpha-fetoprotein, hemopexin, and alpha-2-macroglobulin (A2M) was examined in terms of the serum level and their fuco-fractions. The results indicated that the tests using the multiplexed fuco-biomarkers provided improved discriminatory power between non- hepatocellular carcinoma and hepatocellular carcinoma subjects compared with the alpha-fetoprotein level or fuco-alpha-fetoprotein test alone. The developed method is expected to facilitate the validation of disease-specific glycan biomarker candidates. PMID:25525205

  5. Rapid screening method for quinolone residues in livestock and fishery products using immobilised metal chelate affinity chromatographic clean-up and liquid chromatography-fluorescence detection.

    PubMed

    Takeda, N; Gotoh, M; Matsuoka, T

    2011-09-01

    An efficient LC method was developed for screening the presence of quinolones (QLs)--comprising fluoroquinolones (FQs) and acidic quinolones (AQs)--residues in various livestock and fishery products. Targeted analytes were for nine FQs of marbofloxacin (MAR), ofloxacin (OFL), norfloxacin (NOR), ciprofloxacin (CIP), enrofloxacin (ENR), danofloxacin (DAN), orbifloxacin (ORB), difloxacin (DIF) and sarafloxacin (SAR), and three AQs of oxolinic acid (OXA), nalidixic acid (NAL) and flumequine (FMQ). Samples comprised ten different food products covering five matrices: muscle (cattle, swine and chicken), liver (chicken), raw fish (shrimp and salmon), egg (chicken), and processed food (ham, sausage and fish sausage). This method involved a simple extraction with (1:1) acetonitrile-methanol, a highly selective clean-up with an immobilised metal chelate affinity column charged with Fe(3+), a fast isocratic LC analysis using a short column (20 mm × 4.6 mm, 3 µm) with a mobile phase of (15:85:0.1) methanol/water/formic acid, and fluorescence detection (excitation/emission wavelengths of 295 nm/455 nm for FQs (495 nm for MAR), and 320 nm/365 nm for AQs). Among FQs, pairs of NOR/OFL, ORB/DIF and ENR/DAN were incompletely resolved. A confirmatory LC run with a Mg(2+) containing methanolic mobile phase was also proposed for the samples suspected of being positive. The optimised method gave satisfactory recoveries of 88.5% (56.1-108.6%) and 78.7% (44.1-99.5%) for intra- and inter-day assays with relative standard deviations of 7.2% (0.7-18.4%) and 6.8% (1.4-16.6%), respectively. Limits of quantitation ranged from 0.8 µg kg(-1) (DAN) to 6.5 µg kg(-1) (SAR). This method was successfully employed to analyse 113 real samples and two positive samples were found: fish sausage (CIP 990 µg kg(-1)) and shrimp (ENR 20 µg kg(-1)). PMID:21749230

  6. Profiling of cis-diol-containing nucleosides and ribosylated metabolites by boronate-affinity organic-silica hybrid monolithic capillary liquid chromatography/mass spectrometry.

    PubMed

    Jiang, Han-Peng; Qi, Chu-Bo; Chu, Jie-Mei; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-01-01

    RNA contains a large number of modified nucleosides. In the metabolic re-exchange of RNA, modified nucleosides cannot be recycled and are thus excreted from cells into biological fluids. Determination of endogenous modified nucleosides in biological fluids may serve as non-invasive cancers diagnostic methods. Here we prepared boronate-affinity organic-silica hybrid capillary monolithic column (BOHCMC) that exhibited excellent selectivity toward the cis-diol-containing compounds. We then used the prepared BOHCMC as the on-line solid-phase microextraction (SPME) column and developed an on-line SPME-LC-MS/MS method to comprehensively profile cis-diol-containing nucleosides and ribosylated metabolites in human urine. Forty-five cis-diol-containing nucleosides and ribosylated metabolites were successfully identified in human urine. And five ribose conjugates, for the first time, were identified existence in human urine in the current study. Furthermore, the relative quantification suggested 4 cis-diol-containing compounds (5'-deoxy-5'-methylthioadensine, N(4)-acetylcytidine, 1-ribosyl-N-propionylhistamine and N(2),N(2),7-trimethylguanosine) increased more than 1.5 folds in all the 3 types of examined cancers (lung cancer, colorectal cancer, and nasopharyngeal cancer) compared to healthy controls. The on-line SPME-LC-MS/MS method demonstrates a promising method for the comprehensive profiling of cis-diol-containing ribose conjugates in human urines, which provides an efficient strategy for the identification and discovery of biomarkers and may be used for the screening of cancers. PMID:25585609

  7. Profiling of cis-Diol-containing Nucleosides and Ribosylated Metabolites by Boronate-affinity Organic-silica Hybrid Monolithic Capillary Liquid Chromatography/Mass Spectrometry

    PubMed Central

    Jiang, Han-Peng; Qi, Chu-Bo; Chu, Jie-Mei; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-01-01

    RNA contains a large number of modified nucleosides. In the metabolic re-exchange of RNA, modified nucleosides cannot be recycled and are thus excreted from cells into biological fluids. Determination of endogenous modified nucleosides in biological fluids may serve as non-invasive cancers diagnostic methods. Here we prepared boronate-affinity organic-silica hybrid capillary monolithic column (BOHCMC) that exhibited excellent selectivity toward the cis-diol-containing compounds. We then used the prepared BOHCMC as the on-line solid-phase microextraction (SPME) column and developed an on-line SPME-LC-MS/MS method to comprehensively profile cis-diol-containing nucleosides and ribosylated metabolites in human urine. Forty-five cis-diol-containing nucleosides and ribosylated metabolites were successfully identified in human urine. And five ribose conjugates, for the first time, were identified existence in human urine in the current study. Furthermore, the relative quantification suggested 4 cis-diol-containing compounds (5′-deoxy-5′-methylthioadensine, N4-acetylcytidine, 1-ribosyl-N-propionylhistamine and N2,N2,7-trimethylguanosine) increased more than 1.5 folds in all the 3 types of examined cancers (lung cancer, colorectal cancer, and nasopharyngeal cancer) compared to healthy controls. The on-line SPME-LC-MS/MS method demonstrates a promising method for the comprehensive profiling of cis-diol-containing ribose conjugates in human urines, which provides an efficient strategy for the identification and discovery of biomarkers and may be used for the screening of cancers. PMID:25585609

  8. Immobilized magnetic beads based multi-target affinity selection coupled with high performance liquid chromatography-mass spectrometry for screening anti-diabetic compounds from a Chinese medicine "Tang-Zhi-Qing".

    PubMed

    Tao, Yi; Chen, Zhui; Zhang, Yufeng; Wang, Yi; Cheng, Yiyu

    2013-05-01

    We developed an approach for screening bioactive compounds from botanical drug using multiple target-immobilized magnetic beads coupled with high performance liquid chromatography-mass spectrometry. This novel approach was called magnetic beads based multi-target affinity selection-mass spectrometry (MT-ASMS). It can enrich and identify different types of ligands from mixture extracts. Multiple targets (maltase, invertase, lipase) were immobilized on the magnetic beads by covalent linkage using 1-(3-dimethyl-aminopropyl)-3-ethyl-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as reaction reagents, respectively. The properties of enzyme conjugated magnetic beads were characterized using transmission electron microscopy, X-ray diffractometer and vibration sample magnetometer. Several factors including pH, ion strength, incubation time and temperature were optimized using three known ligands (caffeic acid, ferulic acid, and hesperidin). The established MT-ASMS approach was applied to screening for ligands from a Chinese medicine "Tang-Zhi-Qing", which was used to treat type II diabetes in China. Seven bound compounds were identified via liquid chromatography-mass spectrometry (LC/MS). Five active compounds including 2,3,4,6-tetra-O-galloyl-D-glucose, 1,2,3,4-tetra-O-galloyl-D-glucose, 1,2,3,4,6-penta-O-galloyl-d-glucose, quercetin-3-O-β-D-glucuronide and quercetin-3-O-β-D-glucoside were identified and their activities were validated by conventional inhibitory assay. Our findings suggested that the proposed approach is efficient in screening compounds with multiple activities from extracts of botanical drugs. PMID:23501439

  9. Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography.

    PubMed

    Sriwanthana, B; Island, M D; Maneval, D; Mobley, H L

    1994-11-01

    Proteus mirabilis urease, a nickel metalloenzyme, is essential for the virulence of this species in the urinary tract. Escherichia coli containing cloned structural genes ureA, ureB, and ureC and accessory genes ureD, ureE, ureF, and ureG displays urease activity when cultured in M9 minimal medium. To study the involvement of one of these accessory genes in the synthesis of active urease, deletion mutations were constructed. Cultures of a ureE deletion mutant did not produce an active urease in minimal medium. Urease activity, however, was partially restored by the addition of 5 microM NiCl2 to the medium. The predicted amino acid sequence of UreE, which concludes with seven histidine residues among the last eight C-terminal residues (His-His-His-His-Asp-His-His-His), suggested that UreE may act as a Ni2+ chelator for the urease operon. To exploit this potential metal-binding motif, we attempted to purify UreE from cytoplasmic extracts of E. coli containing cloned urease genes. Soluble protein was loaded onto a nickel-nitrilotriacetic acid column, a metal chelate resin with high affinity for polyhistidine tails, and bound protein was eluted with a 0 to 0.5 M imidazole gradient. A single polypeptide of 20-kDa apparent molecular size, as shown by sodium dodecyl sulfate-10 to 20% polyacrylamide gel electrophoresis, was eluted between 0.25 and 0.4 M imidazole. The N-terminal 10 amino acids of the eluted polypeptide exactly matched the deduced amino acid sequence of P. mirabilis UreE. The molecular size of the native protein was estimated on a Superdex 75 column to be 36 kDa, suggesting that the protein is a dimer. These data suggest that UreE is a Ni(2)+-binding protein that is necessary for synthesis of a catalytically active urease at low Ni(2+) concentrations. PMID:7961442

  10. An SDS-PAGE Examination of Protein Quaternary Structure and Disulfide Bonding for a Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Powers, Jennifer L.; Andrews, Carla S.; St. Antoine, Caroline C.; Jain, Swapan S.; Bevilacqua, Vicky L. H.

    2005-01-01

    Electrophoresis is a valuable tool for biochemists, yet this technique is often not included in biochemistry laboratory curricula owing to time constraints or lack of equipment. Protein structure is also a topic of interest in many disciplines, yet most undergraduate lab experiments focus only on primary structure. In this experiment, students use…

  11. Teaching the Structure of Immunoglobulins by Molecular Visualization and SDS-PAGE Analysis

    ERIC Educational Resources Information Center

    Rižner, Tea Lanišnik

    2014-01-01

    This laboratory class combines molecular visualization and laboratory experimentation to teach the structure of the immunoglobulins (Ig). In the first part of the class, the three-dimensional structures of the human IgG and IgM molecules available through the RCSB PDB database are visualized using freely available software. In the second part, IgG…

  12. Analysis of differentially expressed proteins in colorectal cancer using hydroxyapatite column and SDS-PAGE.

    PubMed

    Lim, Shi-Rou; Gooi, Boon-Hui; Singh, Manjit; Gam, Lay-Harn

    2011-11-01

    Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method. PMID:21863284

  13. Advanced negative detection method comparable to silver stain for SDS-PAGE separated proteins detection.

    PubMed

    Wang, Xu; Hwang, Sun-Young; Cong, Wei-Tao; Jin, Li-Tai; Choi, Jung-Kap

    2016-10-01

    In order to achieve an easy, rapid and sensitive protocol to detect proteins in polyacrylamide gel, an advanced negative detection method comparable to silver stain is described. When a gel was incubated with Phloxine B and followed by the development in acidic solution, the zones where forming protein-dye complex were selectively transparent, unlike opaque gel background. Within 50 min after electrophoresis, down to 0.1-0.4 ng of gel-separated proteins (similar with silver stain) could be observed, without labor-intensive and time-consuming procedure. Comparing with the most common negative stain method, Imidazole-zinc stain, Phloxine B stain has been shown higher sensitivity and distinct contrast between the transparent protein bands/spots and opaque background than those; furthermore, it is no longer necessary to concern about retention time of observation. This technique may provide a sensitive and practical choice for proteomics researches. PMID:27430933

  14. Expression of cold-adapted β-1,3-xylanase as a fusion protein with a ProS2 tag and purification using immobilized metal affinity chromatography with a high concentration of ArgHCl.

    PubMed

    Kudou, Motonori; Okazaki, Fumiyoshi; Asai-Nakashima, Nanami; Ogino, Chiaki; Kondo, Akihiko

    2015-01-01

    Cold-adapted β-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes. PMID:25214227

  15. Virus-like particles from Escherichia Coli-derived untagged papaya ringspot virus capsid protein purified by immobilized metal affinity chromatography enhance the antibody response against a soluble antigen.

    PubMed

    Guerrero-Rodríguez, Jesús; Manuel-Cabrera, Carlos Alberto; Palomino-Hermosillo, Y Apatzingan; Delgado-Guzmán, Paola Guadalupe; Escoto-Delgadillo, Martha; Silva-Rosales, Laura; Herrera-Rodríguez, Sara Elisa; Sánchez-Hernández, Carla; Gutiérrez-Ortega, Abel

    2014-12-01

    There is a growing interest in using virus-like particles (VLPs) as scaffolds for the presentation of antigens of choice to the immune system. In this work, VLPs from papaya ringspot virus capsid protein expressed in Escherichia coli were evaluated as enhancers of antibody response against a soluble antigen. Interestingly, although the capsid protein lacks a histidine tag, its purification by immobilized metal affinity chromatography was achieved. The formation of VLPs was demonstrated by electron microscopy for the first time for this capsid protein. VLPs were enriched by polyethylene glycol precipitation. Additionally, these VLPs were chemically coupled to green fluorescent protein in order to evaluate them as antigen carriers; however, bioconjugate instability was observed. Nonetheless, the adjuvant effect of these VLPs on BALB/c mice was evaluated, using GFP as antigen, resulting in a significant increase in anti-GFP IgG response, particularly, IgG1 class, demonstrating that the VLPs enhance the immune response against the antigen chosen in this study. PMID:25119647

  16. KINETIC STUDIES OF DRUG-PROTEIN INTERACTIONS BY USING PEAK PROFILING AND HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: EXAMINATION OF MULTI-SITE INTERACTIONS OF DRUGS WITH HUMAN SERUM ALBUMIN COLUMNS

    PubMed Central

    Tong, Zenghan; Schiel, John E.; Papastavros, Efthimia; Ohnmacht, Corey M.; Smith, Quentin R.; Hage, David S.

    2010-01-01

    Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (± 0.2) s-1 and 0.67 (± 0.04) s-1 at pH 7.4 and 37 °C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins. PMID:21067755

  17. Magnetic Parkia pendula seed gum as matrix for Concanavalin A lectin immobilization and its application in affinity purification.

    PubMed

    Rêgo, Moacyr J B M; Almeida, Sinara M; Bezerra, Sérgio A; Carvalho Júnior, Luiz B; Beltrão, Eduardo I C

    2014-09-01

    The present work aimed to magnetize Parkia pendula seeds gum and use it as a matrix for Concanavalin A covalent immobilization. This composite was applied in affinity purification of glycoconjugates. Parkia pendula seeds were hydrated and the gum provenient from the supernatant was precipitated and washed with ethanol and dried. The gum was magnetized in co-precipitation using solutions of Fe+2 and Fe+3. Matrix activation was accomplished with NaIO4. Magnetized Parkia pendula seeds gum with covalently immobilized Concanavalin A was used as an affinity matrix for the recognition of bovine serum fetuin glycoprotein. Fetuin elution was carried out with a solution of glucose (300mM) and evaluated through SDS-PAGE. The efficiency of lectin immobilization and fetuin purification were 63% and 14%, respectively. These results indicate that the composite produced is a promising magnetic polysaccharide matrix for lectins immobilization. Thus, such system can be applied for affinity purification allowing an easy recovery by magnetic field. PMID:25140501

  18. Low-affinity transport of FITC-albumin in alveolar type II epithelial cell line RLE-6TN.

    PubMed

    Tagawa, Maki; Yumoto, Ryoko; Oda, Keisuke; Nagai, Junya; Takano, Mikihisa

    2008-01-01

    FITC-albumin uptake by cultured alveolar type II epithelial cells, RLE-6TN, is mediated by high- and low-affinity transport systems. In this study, characteristics of the low-affinity transport system were evaluated. The uptake of FITC-albumin was time and temperature dependent and was inhibited by metabolic inhibitors and bafilomycin A1. Confocal laser scanning microscopic analysis showed punctate localization of the fluorescence in the cells, which was partly localized in lysosomes. FITC-albumin taken up by the cells gradually degraded over time, as shown by fluoroimage analyzer after SDS-PAGE. The uptake of FITC-albumin by RLE-6TN cells was not inhibited by caveolae-mediated endocytosis inhibitors such as nystatin, but was inhibited by clathrin-mediated endocytosis inhibitors such as phenylarsine oxide. The uptake was also inhibited by potassium depletion and hypertonicity, conditions known to inhibit clathrin-mediated endocytosis. In addition, macropinocytosis inhibitors such as 5-(N-ethyl-N-isopropyl) amiloride inhibited the uptake. These results indicate that the low-affinity transport of FITC-albumin in RLE-6TN cells is at least in part mediated by clathrin-mediated endocytosis, but not by caveolae-mediated endocytosis. Possible involvement of macropinocytosis was also suggested. PMID:18974609

  19. Affinity purification of aprotinin from bovine lung.

    PubMed

    Xin, Yu; Liu, Lanhua; Chen, Beizhan; Zhang, Ling; Tong, Yanjun

    2015-05-01

    An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg. PMID:25677462

  20. Affinity chromatography of immobilized actin and myosin.

    PubMed Central

    Bottomley, R C; Trayer, I P

    1975-01-01

    Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder. PMID:241335

  1. Specific high-affinity binding of fatty acids to epidermal cytosolic proteins

    SciTech Connect

    Raza, H.; Chung, W.L.; Mukhtar, H. )

    1991-08-01

    Cytosol from rat, mouse, and human skin or rat epidermis was incubated with (3H)arachidonic acid, (14C)retinoic acid, (14C)oleic acid, (3H)leukotriene A4, (3H)prostaglandin E2 (PGE2) or (3H) 15-hydroxyeicosatetraenoic acid (15-HETE), and protein-bound ligands were separated using Lipidex-1000 at 4C to assess the binding specificity. The binding of oleic acid and arachidonic acid with rat epidermal cytosol was rapid, saturable, and reversible. Binding of oleic acid was competed out with the simultaneous addition of other ligands and found to be in the following order: arachidonic acid greater than oleic acid greater than linoleic acid greater than lauric acid greater than leukotriene A4 greater than 15-HETE = PGE1 greater than PGE2 = PGF2. Scatchard analysis of the binding with arachidonic acid, oleic acid, and retinoic acid revealed high-affinity binding sites with the dissociation constant in the nM range. SDS-PAGE analysis of the oleic acid-bound epidermal cytosolic protein(s) revealed maximum binding at the 14.5 kDa region. The presence of the fatty acid-binding protein in epidermal cytosol and its binding to fatty acids and retinoic acid may be of significance both in the trafficking and the metabolism of fatty acids and retinoids across the skin.

  2. Purification of a thermostable chitinase from Bacillus cereus by chitin affinity and its application in microbial community changes in soil.

    PubMed

    Liang, Tzu-Wen; Hsieh, Tung-Yen; Wang, San-Lang

    2014-06-01

    A thermostable chitinase was purified by chitin affinity from the culture supernatant of Bacillus cereus TKU028 with shrimp head powder (SHP) as the sole carbon/nitrogen source. TKU028 chitinase was purified using a one-step affinity adsorbent system, and the molecular mass of TKU028 chitinase (approximately 40 kDa) was then determined using SDS-PAGE. The enzyme was stable for 60 min at temperatures below 60 °C and stable over a broad pH range of 4-9 for 60 min. In addition, the temporal changes of a bacterial community in mangrove river sediment of the Tamsui River with added SHP were also analysed by PCR-denaturing gradient gel electrophoresis to investigate the effects of B. cereus TKU028 on the degradation of SHP. The 6-week incubation sample of SHP and B. cereus TKU028-amended mangrove river sediment displayed the highest amount of biomass, reducing sugar and total sugar, and some variance of bacterial community composition existed in the soils. PMID:24342954

  3. Crataeva tapia bark lectin is an affinity adsorbent and insecticidal agent.

    PubMed

    Araújo, Regina Maria Sousa de; Ferreira, Rodrigo da Silva; Napoleão, Thiago Henrique; Carneiro-da-Cunha, Maria das Graças; Coelho, Luana Cassandra Breitenbach Barroso; Correia, Maria Tereza Dos Santos; Oliva, Maria Luiza Vilela; Paiva, Patrícia Maria Guedes

    2012-02-01

    Hemagglutinating activity has been associated to presence of lectin, carbohydrate-binding proteins. In this work Crataeva tapia bark lectin (CrataBL) was purified in milligram quantities (28 mg per g of bark) by ion exchange chromatography. The lectin was thermo-stable, ion-independent and N-terminal sequence analysis demonstrated similarity with miraculin and miraculin-like proteins (plant defensive proteins). Glycosylated nature of CrataBL was revealed using glycoprotein staining (periodic acid-Schiff's reagent), positive for polypeptides of apparent molecular masses 21 and 40 kDa on SDS-PAGE. Gel diffusion assay showed that glucose/mannose isolectins from Cratylia mollis recognized CrataBL glycan moiety. CrataBL hemagglutinating activity was inhibited by glycoproteins and CrataBL immobilized on cyanogen bromide-activated sepharose 4B (1 mL) bound 0.54 mg of glycoprotein (casein, fetuin and ovalbumin) per cycle. CrataBL was an insecticide agent against Nasutitermes corniger workers (termite that attack woods) with LC₅₀ of 0.475 mg mL⁻¹ for 6 days. PMID:22195573

  4. Confirmation of a blocked amino terminus of sulfhydryl oxidase

    SciTech Connect

    Janolino, V.G.; Morrison-Rowe, S.J.; Swaisgood, H.E. )

    1990-09-01

    The isolation of sulfhydryl oxidase from bovine milk in a suitably pure form for sequencing was carried out by transient covalent affinity chromatography of diafiltered whey using cysteinylsuccinamidopropyl-glass as matrix. The glutathione-eluted proteins were separated by SDS-PAGE. By radiolabeling the affinity chromatography-purified enzyme with ({sup 14}C)iodoacetate before subjecting to SDS-PAGE, the sulfhydryl oxidase band was identified, because sulfhydryl oxidase is known to be inactivated by alkylation of one sulfhydryl group per mole. The results confirmed that sulfhydryl oxidase corresponds to the 85 ({plus minus} 5)-kDa band observed on SDS-PAGE. The protein band corresponding to radiolabeled sulfhydryl oxidase was recovered from SDS-PAGE gels by electrophoretic elution and by electroblotting on polyvinylidene difluoride membrane and subjected to gas phase sequencing. Precautions were taken during electrophoretic elution to prevent reactions that result in N-terminal blocking. Both methods of protein recovery yielded negative results when subjected to sequence analysis indicating that the N-terminus of sulfhydryl oxidase is blocked.

  5. Protein quantitation using various modes of high performance liquid chromatography.

    PubMed

    Grotefend, Sandra; Kaminski, Lukas; Wroblewitz, Stefanie; Deeb, Sami El; Kühn, Nancy; Reichl, Stephan; Limberger, Markus; Watt, Steven; Wätzig, Hermann

    2012-12-01

    Pharmaceuticals based on proteins (biologicals), such as monoclonal antibodies (mAb), attain more and more relevance since they were established as potent drugs in anticancer therapy or for the treatment of autoimmune based diseases. Due to their high efficiency it is essential to have accurate and precise methods for protein quantitation and the detection of protein aggregates, which in some cases may lead to adverse effects after application. Selectivity and precision of traditional protein quantification methods such as the Bradford assay or SDS-PAGE are insufficient for quality control (QC) purposes. In this work several HPLC separation modes, which can significantly improve these important parameters, were compared for their application in this field. High performance size exclusion (HP-SEC), strong anion exchange (SAX), weak cation exchange (WCX) as well as reversed phase chromatography are all already successfully applied in protein analysis. Good precision (SEC: <1.9%, SAX: <5%, RP: <2% and WCX: <3.5% - RSD% for peak areas day-to-day), high selectivity and low quantitation limits (<15μg/ml) for the model proteins ovalbumin, myoglobin and bovine serum albumin (BSA), respectively cytochrome c and lysozyme in the cation exchange mode, could be achieved. Consecutively, the four separation modes were compared to each other and to electrophoretic techniques in terms of precision, selectivity, analysis time, effort of sample and mobile phase preparation as well as separating capacity. Moreover, the analysis of an IgG1-type antibody was included in this study. PMID:22980318

  6. Aptamers in Affinity Separations: Stationary Separation

    NASA Astrophysics Data System (ADS)

    Ravelet, Corinne; Peyrin, Eric

    The use of DNA or RNA aptamers as tools in analytical chemistry is a very promising field of research because of their capabilities to bind specifically the target molecules with an affinity similar to that of antibodies. Notably, they appear to be of great interest as target-specific ligands for the separation and capture of various analytes in affinity chromatography and related affinity-based methods such as magnetic bead technology. In this chapter, the recent developments of these aptamer-based separation/capture approaches are addressed.

  7. Affinity capture of (Arg sup 8 )vasopressin-receptor complex using immobilized antisense peptide

    SciTech Connect

    Feng Xian Lu; Aiyar, N.; Chaiken, I. )

    1991-05-01

    Solubilized noncovalent complexes of (Arg{sup 8})-vasopressin (AVP) with receptor proteins from rat liver membranes were isolated by selective binding to silica-immobilized antisense (AS) peptide. The affinity chromatographic support was prepared with a chemically synthesized AS peptide whose sequence is encoded by the AS DNA corresponding to the 20 amino-terminal residues of the AVP bovine neurophysin II biosynthetic precursor (pro-AVP/BNPII-(20-1)), region that includes the AVP sequence at residues 1-9. The AS peptide-AVP interaction mechanism hypothesized, contact by hydropathic complementarity at multiple sites along the peptide chains, led to the prediction that AVP bound to its receptor would still have enough free surface to interact with immobilized AS peptide. To test this prediction of a three-way interaction, ({sup 3}H)AVP-receptor was obtained as a solubilized, partially purified fraction from rat liver membrane. Covalently crosslinked ({sup 3}H)AVP complex also was bound to the AS peptide column; binding was blocked by competition with unlabeled AVP in the elution buffer. Since the AVP-linked 31- and 38-kDa proteins have the same apparent molecular mass on SDS/PAGE as found previously by photoaffinity labeling, the authors conclude that the AS peptide column has affinity-captured AVP-receptor complexes. The 15-kDa protein appears to be an active AVP-receptor fragment of one or both of the larger proteins. It is generally concluded that immobilized AS peptides may be useful to isolate peptide and protein receptor complexes in other systems as well.

  8. Metal-affinity separations: A new dimension in protein processing

    SciTech Connect

    Arnold, F.H. )

    1991-02-01

    Rapid growth in the preparative and high-resolution analytical applications of metal-affinity chromatography demonstrate the appeal of metal recognition as a basis for protein separations. Stable, inexpensive chelated metals effectively mimic biospecific interactions, providing selective ligands for protein binding. This article reviews recent progress in understanding the mechanisms of metal-protein recognition that underlie metal-affinity separations. Also discussed are schemes for integrating metal-affinity purifications into the expression and bioprocessing of recombinant proteins. Promising future developments include new metal-affinity processes for analytical and preparative-scale separations and a range of techniques for enhancing the selectivity of metal-affinity separations.

  9. Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand

    PubMed Central

    2013-01-01

    Background Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production. Results A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris–HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE. Conclusion A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum. PMID:24341315

  10. The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK

    SciTech Connect

    Cauvin, A.; Buscail, L.; Gourlet, P.; De Neef, P.; Gossen, D.; Arimura, A.; Miyata, A.; Coy, D.H.; Robberecht, P.; Christophe, J. )

    1990-07-01

    We investigated the ability of two forms of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP-38, the 38 amino acid peptide isolated from ovine hypothalamus, and PACAP-27, a shorter N-terminal (1-27) amidated version) to interact with specific receptors in membranes from the human neuroblastoma cell line NB-OK. ({sup 125}I)PACAP-27 bound rapidly and specifically to one class of high affinity sites (Kd 0.5 nM). VIP inhibited ({sup 125}I)PACAP-27 binding 300- to 1000-fold less potently than PACAP-27 and PACAP-38. One microM PHI prevented tracer binding only partially and secretin, glucagon and GRF(1-29)NH2 were ineffective in this respect. PACAP-27 and PACAP-38 stimulated adenylate cyclase activity dose dependently and with similar efficacy (Kact 0.2-0.3 nM), this activation being compatible with the occupancy of specific high affinity PACAP receptor. VIP was markedly less potent and less efficient on this enzyme than PACAP. Chemical cross-linking of ({sup 125}I)PACAP-27 followed by SDS-PAGE and autoradiography revealed specific cross-linking with a 68 kDa protein.

  11. A simplified method for purification of annexin V from human placenta.

    PubMed

    Poghosyan, G G; Melkonyan, V Z; Mikaelyan, M V; Gasparyan, V K

    2003-08-01

    A simplified procedure for purification of annexin V from human placenta was developed. At first, the protein was separated from other proteins in membrane bound form in the presence of Ca2+, then was extracted with EDTA and purified by affinity chromatography on PAAG-immobilized phosphatidylserine. The purified protein gave a single band with a molecular weight of 35,000 in SDS-PAGE. PMID:12916812

  12. Identification of protein interacting partners using tandem affinity purification.

    PubMed

    Bailey, Dalan; Urena, Luis; Thorne, Lucy; Goodfellow, Ian

    2012-01-01

    proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function. PMID:22395237

  13. Applying Chromatography.

    ERIC Educational Resources Information Center

    Klein, Jessie W.; Patev, Paul

    1998-01-01

    Presents three experiments to introduce students to different kinds of chromatography: (1) paper chromatography; (2) gel filtration chromatography; and (3) reverse-phase liquid chromatography. Written in the form of a laboratory manual, explanations of each of the techniques, materials needed, procedures, and a glossary are included. (PVD)

  14. Antihemorrhagin in the blood serum of king cobra (Ophiophagus hannah): purification and characterization.

    PubMed

    Chanhome, Lawan; Khow, Orawan; Omori-Satoh, Tamotsu; Sitprija, Visith

    2003-06-01

    King cobra (Ophiophagus hannah) serum was found to possess antihemorrhagic activity against king cobra hemorrhagin. The activity was stronger than that in commercial king cobra antivenom. An antihemorrhagin has been purified by ion exchange chromatography, affinity chromatography and gel filtration with a 22-fold purification and an overall yield of 12% of the total antihemorrhagic activity contained in crude serum. The purified antihemorrhagin was homogeneous in disc-PAGE and SDS-PAGE. Its apparent molecular weight determined by SDS-PAGE was 120 kDa. The antihemorrhagin was also active against other hemorrhagic snake venoms obtained in Thailand and Japan such as Calloselasma rhodostoma, Trimeresurus albolabris, Trimeresurus macrops and Trimeresurus flavoviridis (Japanese Habu). It inhibited the proteolytic activity of king cobra venom. It is an acid- and thermolabile protein and does not form precipitin lines against king cobra venom. PMID:12875876

  15. Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA).

    PubMed

    Miller, Daniel E; Patel, Zubin H; Lu, Xiaoming; Lynch, Arthur T; Weirauch, Matthew T; Kottyan, Leah C

    2016-01-01

    Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype. PMID:27585267

  16. A sub-population of keratan sulphates derived from bovine articular cartilage is capped with alpha(2-6)-linked N-acetylneuraminic acid residues. Affinity chromatography using immobilized Sambucus nigra lectin and characterization using 1H n.m.r. spectroscopy.

    PubMed Central

    Tai, G H; Morris, H G; Brown, G M; Huckerby, T N; Nieduszynski, I A

    1992-01-01

    Alkaline borohydride-reduced keratan sulphate (KS) chains derived from bovine femoral head cartilage were fractionated by lectin affinity chromatography with Sambucus nigra agglutinin (SNA) into binding and non-binding populations. Analysis of the SNA-binding and non-binding KS chains using 600 MHz 1H n.m.r. spectroscopy showed that the former population contained alpha(2-6)-N-acetylneuraminic acid residues and the latter contained primarily alpha(2-3)-N-acetylneuraminic acid residues as chain terminators. Both populations contained a similar proportion of alpha(2-3)-N-acetylneuraminic acid residues within their protein-linkage regions, and similar sulphation and fucosylation levels. Analysis of these two fractions by gel-permeation chromatography (g.p.c.) on a TSK-30 XL column showed them to have the same size distributions. It was concluded from the n.m.r. spectra and g.p.c. data that the populations differed primarily in the mode of linkage of the chain-terminating sialic acids. PMID:1520274

  17. Purification of selenoprotein P from human plasma using immunoaffinity chromatography

    SciTech Connect

    Aakesson, B.; Bellew, T.; Burk, R.F. )

    1991-03-11

    Selenoprotein P was purified from rat plasma using immunoaffinity chromatography. The same approach was used with human plasma. HepG2 cells were labeled with {sup 75}Se. The labeled medium, containing proteins secreted by the cells, was added to human plasma and the {sup 75}Se was used as a marker for {gt}1,000-fold purification of the major {sup 75}Se-containing protein. This material was used to produce 2 monoclonal antibodies. In a competitive assay, human plasma, but not plasma from 5 other species, inhibited binding of {sup 75}Se by these 2 antibodies. The antibodies were coupled to agarose and columns were made. Human plasma was processed in 2 steps. Step 1 was an antibody column and step 2 was a heparin-agarose column. SDS-PAGE demonstrated bands at 61 and 55 kDa. Both bands stained with PAS. Amino acid analysis of carboxymethylated material indicated that selenocysteine was {gt}1% of the total amino acids. N-terminal sequencing revealed a strong similarity to rat selenoprotein P. Immunodepleted human plasma and control plasma were chromatographed on Sephacryl S200 and selenium was measured in the eluted fractions. Immunodepletion removed one-third of the selenium. The elution pattern of control plasma revealed a broad peak of selenium just ahead of and including the albumin peak. Most of this peak was absent from the immunodepleted serum and a graph of the difference between the 2 chromatograms was a single peak of selenium well separated from the albumin peak.

  18. Production and purification of human papillomavirus type 33 L1 virus-like particles from Spodoptera frugiperda 9 cells using two-step column chromatography.

    PubMed

    Baek, Jin-Oh; Seo, Jeong-Woo; Kim, Ik-Hwan; Kim, Chul Ho

    2011-02-01

    The major capsid protein L1 of human papillomavirus (HPV) is essential in construction of recombinant antigen vaccines against cervical cancer. HPV type 33 accounts for about 10% of all HPV infections in Asia. The gene encoding the major capsid protein L1 of the high-risk HPV type 33 was isolated from a Korean patient and expressed in Sf-9 insect cells using a baculovirus expression system. HPV33 L1 protein was isolated by two-step chromatographic purification using strong-cation exchange and ceramic hydroxyapatite chromatography. Strong-cation-exchange chromatography was performed to achieve initial purification of HPV33 L1 and to remove most contaminating proteins, and secondary ceramic hydroxyapatite chromatography yielded pure HPV33 L1 virus-like particles (VLPs). Ceramic hydroxyapatite columns are particularly useful in the purification of antibodies, antigens, human viruses, and VLPs, and we thus used this system. The expression of HPV L1 protein in Sf-9 cells was examined by SDS-PAGE, Western-blotting, and ELISA analyses, and the data showed that HPV33 L1 VLPs were determined to > 98% purity and 58.7% recovery by a quantitative immuno-ELISA assay. Transmission electron microscopy analysis revealed that the HPV VLPs were approximately 50-60 nm in diameter and created by self-assembly of HPV L1 protein. The efficient and simple purification process described here should be useful in production of a cervical cancer vaccine. PMID:20716445

  19. High affinity of lead for fetal haemoglobin.

    PubMed Central

    Ong, C N; Lee, W R

    1980-01-01

    In-vitro experiments using 203Pb were performed to identify lead-binding components in human haemoglobin. Sephadex A-50 ion-exchange chromatography of haemolysate showed that different types of haemoglobin had different affinities for lead. For the haemolysate from adults, lead was present in both Hb A (alpha 2 beta 2) and Hb A2 (alpha 2 delta 2), whereas, in the haemolysate from new-born infants, the haemoglobin of fetal origin, Hb F (alpha 2 gamma 2) showed a much greater affinity for 203Pb than the adult haemoglobin Hb A (alpha 2 beta 2), obtained from maternal blood. Analysis of the 203 Pb-labelled haemoglobin suggested that about 82% of 203Pb was in the globin polypeptide. Further analysis with carboxylmethyl (CM) cellulose chromatography indicated that the gamma globin of fetal origin had a higher affinity for 203Pb than the beta globin, whereas alpha globin appeared to be unimportant in lead binding. The results of the different affinities for lead of different Hb types are discussed with regard to the effect of lead upon haemoglobin synthesis. PMID:6158989

  20. A Method to Site-Specifically Identify and Quantitate Carbonyl End Products of Protein Oxidation Using Oxidation-Dependent Element Coded Affinity Tags (O-ECAT) and NanoLiquid Chromatography Fourier Transform Mass Spectrometry

    SciTech Connect

    Lee, S; Young, N L; Whetstone, P A; Cheal, S M; Benner, W H; Lebrilla, C B; Meares, C F

    2005-08-25

    Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1, 4, 7, 10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. After proteolysis, the resulting AOD-tagged peptides are affinity purified, and analyzed by nanoLC-FTICR-MS, which provides high specificity in extracting co-eluting AOD mass pairs with a unique mass difference and affords relative quantitation based on isotopic ratios. Using this methodology, we have mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities towards oxidation independent of amino acid residue. We expect to extend

  1. Ferromagnetic levan composite: an affinity matrix to purify lectin.

    PubMed

    Angeli, Renata; da Paz, Nathalia V N; Maciel, Jackeline C; Araújo, Flávia F B; Paiva, Patrícia M G; Calazans, Glícia M T; Valente, Ana Paula; Almeida, Fábio C L; Coelho, Luana C B B; Carvalho, Luiz B; Silva, Maria da Paz C; dos Santos Correia, Maria Tereza

    2009-01-01

    A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A) and Cratylia mollis (Cramoll 1 and Cramoll 1, 4) did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column. PMID:19547713

  2. Ferromagnetic Levan Composite: An Affinity Matrix to Purify Lectin

    PubMed Central

    Angeli, Renata; da Paz, Nathalia V. N.; Maciel, Jackeline C.; Araújo, Flávia F. B.; Paiva, Patrícia M. G.; Calazans, Glícia M. T.; Valente, Ana Paula; Almeida, Fábio C. L.; Coelho, Luana C. B. B.; Carvalho, Luiz B.; Silva, Maria da Paz C.; dos Santos Correia, Maria Tereza

    2009-01-01

    A simple and inexpensive procedure used magnetite and levan to synthesize a composite recovered by a magnetic field. Lectins from Canavalia ensiformis (Con A) and Cratylia mollis (Cramoll 1 and Cramoll 1, 4) did bind specifically to composite. The magnetic property of derivative favored washing out contaminating proteins and recovery of pure lectins with glucose elution. Cramoll 1 was purified by this affinity binding procedure in two steps instead of a previous three-step protocol with ammonium sulfate fractionation, affinity chromatography on Sephadex G-75, and ion exchange chromatography through a CM-cellulose column. PMID:19547713

  3. Changes in circulating immune complexes in tumour patient serum after in vitro or ex vivo affinity chromatography of blood plasma or whole blood over immunoglobulin-binding staphylococcal protein A-Sepharose.

    PubMed

    Håkansson, L; Hed, J; Baldetorp, L; Eneström, S; Jonsson, S; Liedén, G

    1984-01-01

    Circulating immune complexes (CIC) were determined in tumour patient sera using three methods. One is based on PEG-precipitation, one on C1q-reactivity, and one on protein A-reactivity. About 25-30% of the sera were positive in at least one of the tests. Incubation of serum with protein A-Sepharose in vitro removed PEG-precipitable CIC from most sera, whereas C1q-reactive CICs had a much lower affinity to protein A. The protein A-reactive complexes showed considerable variation in their binding to protein A-Sepharose, and in some sera the amount of these CICs was actually increased. Similar changes in protein A-reactive CIC were also found during ex vivo treatment of tumour patients with immune adsorption. It is proposed that the binding of immune complexes to protein A can result in remodelling of protein A itself. Results from ultracentrifugation and fractionated PEG-precipitation support this hypothesis. PMID:6365797

  4. A simple one pot purification of bacterial amylase from fermented broth based on affinity toward starch-functionalized magnetic nanoparticle.

    PubMed

    Paul, Tanima; Chatterjee, Saptarshi; Bandyopadhyay, Arghya; Chattopadhyay, Dwiptirtha; Basu, Semanti; Sarkar, Keka

    2015-08-18

    Surface-functionalized adsorbant particles in combination with magnetic separation techniques have received considerable attention in recent years. Selective manipulation on such magnetic nanoparticles permits separation with high affinity in the presence of other suspended solids. Amylase is used extensively in food and allied industries. Purification of amylase from bacterial sources is a matter of concern because most of the industrial need for amylase is met by microbial sources. Here we report a simple, cost-effective, one-pot purification technique for bacterial amylase directly from fermented broth of Bacillus megaterium utilizing starch-coated superparamagnetic iron oxide nanoparticles (SPION). SPION was prepared by co-precipitation method and then functionalized by starch coating. The synthesized nanoparticles were characterized by transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID, zeta potential, and ultraviolet-visible (UV-vis) and Fourier-transform infrared (FTIR) spectroscopy. The starch-coated nanoparticles efficiently purified amylase from bacterial fermented broth with 93.22% recovery and 12.57-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the purified amylase was 67 kD, and native gel showed the retention of amylase activity even after purification. Optimum pH and temperature of the purified amylase were 7 and 50°C, respectively, and it was stable over a range of 20°C to 50°C. Hence, an improved one-pot bacterial amylase purification method was developed using starch-coated SPION. PMID:24840788

  5. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture. PMID:27473483

  6. Preparation of immunoglobulin Y (IgY) against lipopolysaccharide using gel chromatography from the yolks of eggs laid by immunized hens.

    PubMed

    Ma, Siyuan; Zhang, Yaping

    2010-10-01

    The objective is to prevent and treat injuries caused by lipopolysaccharide (LPS) from gram negative bacteria in animals and humans, we produced antibodies against LPS from egg yolk. LPS from E. coli (O111:B4) mixed with Freund's Adjuvant was used as the immunogen to immunize Roman hens. Immunized eggs were collected, and immunoglobulin Y (IgY) was purified using a water solution, salt precipitation and gel chromatography. The molecular weight and purity were determined by SDS-PAGE, the antibody titer by noncompetitive enzyme-linked immunosorbent assay (ELISA) and antibody activity against LPS by the mortality of mice intraperitoneally injected with LPS or LPS-IgY solutions. IgY against LPS showed two protein bands at 68 and 26 kDa on the gel; the antibody titer was almost 1:25,600. After incubation with LPS, IgY decreased the mortality of mice challenged with LPS. This study provided an efficient way to produce high-titer egg yolk antibodies, which could attenuate lethal effects of LPS, by immunizing hens. Furthermore, the LPS antibody was purified well using a water solution, salting-out and gel chromatography. PMID:20798981

  7. Isolation and Purification of Water Soluble Proteins from Ginger Root (Zingiber officinale) by Two Dimensional Liquid Chromatography

    PubMed Central

    Sandovall, A.O.; Andrews, K.; Wahab, A.; Choudhary, M.I.; Ahmed, A.

    2014-01-01

    The RI-INBRE Centralized Core Facility was established in 2003 and participates annually in Undergraduate Summer Research Program. It provides students hands on research experience in key technologies in biomedical sciences. We present here the isolation and purification of water soluble proteins from ginger, a rhizome of the plant, Zingiber officinale. It is an important ingredient of species used in traditional South Asian cuisines. In Indian, Pakistani and Chinese folk medicine, ginger is used for gastro-intestinal disorders, nausea, vomiting, inflammatory diseases, muscle and joint pain. Limited studies have been reported on the bioactive proteins from ginger extract. The water soluble proteins were extracted from ginger root and successfully purified to homogeneity by using two-dimensional liquid chromatography (FPLC/RP-HPLC) approach. The ginger root was washed with distilled water; skin removed and then emulsified using an electric blender. Sample was stirred for four days at 4°C with and without protease inhibitor. Purification of a 42kDa protein was achieved by employing gel filtration, ion-exchange and reversed phase HPLC. The homogeneity of the protein was confirmed by SDS-PAGE gel electrophoresis and MALDI-TOF mass spectrometry. Future work will be conducted on the protein characterization using mass spectrometry and Edman protein sequencing. Supported by grant 5P20GM103430 from the National Institute of General Medical Sciences, NIH, USA.

  8. Gas Chromatography.

    ERIC Educational Resources Information Center

    Karasek, Francis W.; And Others

    1984-01-01

    This review covers fundamental developments in gas chromatography during 1982 and 1983. Literature is considered under these headings: columns; liguid phases; solid supports; sorption processes and solvents; open tubular column gas chromatography; instrumentation; high-resolution columns and applications; other techniques; qualitative and…

  9. Protein affinity map of chemical space.

    PubMed

    Kauvar, L M; Villar, H O; Sportsman, J R; Higgins, D L; Schmidt, D E

    1998-09-11

    Affinity fingerprinting is a quantitative method for mapping chemical space based on binding preferences of compounds for a reference panel of proteins. An effective reference panel of <20 proteins can be empirically selected which shows differential interaction with nearly all compounds. By using this map to iteratively sample the chemical space, identification of active ligands from a library of 30,000 candidate compounds has been accomplished for a wide spectrum of specific protein targets. In each case, <200 compounds were directly assayed against the target. Further, analysis of the fingerprint database suggests a strategy for effective selection of affinity chromatography ligands and scaffolds for combinatorial chemistry. With such a system, the large numbers of potential therapeutic targets emerging from genome research can be categorized according to ligand binding properties, complementing sequence based classification. PMID:9792501

  10. Gas Chromatography.

    ERIC Educational Resources Information Center

    Cram, Stuart P.; And Others

    1980-01-01

    Selects fundamental developments in theory, methodology, and instrumentation in gas chromatography (GC). A special section reviews GC in the People's Republic of China. Over 1,000 references are cited. (CS)

  11. Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10 μm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry.

    PubMed

    Wang, Dongdong; Hincapie, Marina; Rejtar, Tomas; Karger, Barry L

    2011-03-15

    Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ∼100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace

  12. Process for recovering metals from solution utilizing metalloprotein affinity chromatography

    SciTech Connect

    Spears, D.R.; Vincent, J.B.

    1993-11-29

    The invention relates generally to a process for recovering metals from an aqueous metal-bearing solution and, more particularly, to a process which utilizes metalloproteins immobilized on an insoluble support to remove metal ions such as the main group, transition, lanthanide, and actinide ions from the aqueous metal-ion bearing solution.

  13. Analysis of biomolecular interactions using affinity microcolumns: A review

    PubMed Central

    Zheng, Xiwei; Li, Zhao; Beeram, Sandya; Podariu, Maria; Matsuda, Ryan; Pfaunmiller, Erika L.; White, Christopher J.; Carter, NaTasha; Hage, David S.

    2014-01-01

    Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods. PMID:24572459

  14. Analysis of biomolecular interactions using affinity microcolumns: a review.

    PubMed

    Zheng, Xiwei; Li, Zhao; Beeram, Sandya; Podariu, Maria; Matsuda, Ryan; Pfaunmiller, Erika L; White, Christopher J; Carter, NaTasha; Hage, David S

    2014-10-01

    Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods. PMID:24572459

  15. FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici.

    PubMed

    Ben M'Barek, Sarrah; Cordewener, Jan H G; Tabib Ghaffary, Seyed M; van der Lee, Theo A J; Liu, Zhaohui; Mirzadi Gohari, Amir; Mehrabi, Rahim; America, Antoine H P; Robert, Olivier; Friesen, Timothy L; Hamza, Sonia; Stergiopoulos, Ioannis; de Wit, Pierre J G M; Kema, Gerrit H J

    2015-06-01

    Culture filtrates (CFs) of the fungal wheat pathogen Zymoseptoria tritici were assayed for necrosis-inducing activity after infiltration in leaves of various wheat cultivars. Active fractions were partially purified and characterized. The necrosis-inducing factors in CFs are proteinaceous, heat stable and their necrosis-inducing activity is temperature and light dependent. The in planta activity of CFs was tested by a time series of proteinase K (PK) co-infiltrations, which was unable to affect activity 30min after CF infiltrations. This suggests that the necrosis inducing proteins (NIPs) are either absent from the apoplast and likely actively transported into mesophyll cells or protected from the protease by association with a receptor. Alternatively, plant cell death signaling pathways might be fully engaged during the first 30min and cannot be reversed even after PK treatment. Further fractionation of the CFs with the highest necrosis-inducing activity involved fast performance liquid chromatography, SDS-PAGE and mass spectrometry. This revealed that most of the proteins present in the fractions have not been described before. The two most prominent ZtNIP encoding candidates were heterologously expressed in Pichia pastoris and subsequent infiltration assays showed their differential activity in a range of wheat cultivars. PMID:26092790

  16. An efficient method for purification of nonspecific lipid transfer protein-1 from rice seeds using kiwifruit actinidin proteolysis and ion exchange chromatography.

    PubMed

    Ghobadi, Sirous; Yousefi, Fakhroddin; Khademi, Fatemeh; Padidar, Samira; Mostafaie, Ali

    2012-11-01

    Plant nonspecific lipid transfer proteins are small basic proteins that transport phospholipids between membranes and are subdivided into two subfamilies, nsLTP(1) (9 kDa) and nsLTP(2) (7 kDa). LTPs have potential application in the defense reactions against pathogens and the drug delivery systems. Many efforts have been made for purification of different nsLTPs from various plants; however, most of them used successive purification procedures. We have developed a relatively simple and efficient method for the purification of rice nsLTP(1), based on the proteolytic activity of kiwifruit actinidin on the rice seed extract and one-step chromatographic procedure on a CM-Sepharose column. The purity of protein was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The isolated LTP(1) migrated as a homogenous polypeptide with molecular mass of 9 kDa that confirms the efficiency of actinidin on the digestion of major contaminations present in the rice seed extract without any harmful effect on the LTP(1). The advantages of using proteolytic activity of actinidin in purifying rice LTP(1) includes the reduced separation time allowing the purification of LTP(1) in one-step chromatographic procedure, low costing, high efficiency, and the relative simplicity of the method. PMID:22987614

  17. Affinity separation in magnetically stabilized fluidized beds: synthesis and performance of packing materials

    SciTech Connect

    Lochmueller, C.H.; Wigman, L.S.

    1987-11-01

    A magnetically stabilized fluidized-bed separator designed to test the use of pellicular, ferromagnetic affinity chromatography packing materials has been developed. A wire wound solenoid was used to produce the magnetic field. The ferromagnetic packing material is comprised of a magnetite-containing, polyurethane gel coated onto polystyrene beads. The gel contains free carboxyl groups. These were carbodiimide-coupled to soy trypsin inhibitor and the material used for trypsin purification. Narrow-band affinity chromatography was carried out in packed-bed, fluidized-bed, and magnetically stabilized, fluidized-bed separators. Pressure drop, capacity, dilution, and peak asymmetry were evaluated for each type of separator. The three types provide comparable efficiency but the fluidized separators exhibit a much lower pressure drop. As might be expected, fluidized-bed separators perform well for affinity chromatography (large k') but poorly for size exclusion chromatography.

  18. Local Affinity Release.

    PubMed

    Delplace, Vianney; Obermeyer, Jaclyn; Shoichet, Molly S

    2016-07-26

    The use of hydrogels for therapeutic delivery is a burgeoning area of investigation. These water-swollen polymer matrices are ideal platforms for localized drug delivery that can be further combined with specific ligands or nanotechnologies to advance the controlled release of small-molecule drugs and proteins. Due to the advantage of hydrophobic, electrostatic, or specific extracellular matrix interactions, affinity-based strategies can overcome burst release and challenges associated with encapsulation. Future studies will provide innovative binding tools, truly stimuli-responsive systems, and original combinations of emerging technologies to control the release of therapeutics spatially and temporally. Local drug delivery can be achieved by directly injecting a therapeutic to its site of action and is advantageous because off-target effects associated with systemic delivery can be minimized. For prolonged benefit, a vehicle that provides sustained drug release is required. Hydrogels are versatile platforms for localized drug release, owing to the large library of biocompatible building blocks from which they can be formed. Injectable hydrogel formulations that gel quickly in situ and provide sustained release of therapeutics are particularly advantageous to minimize invasiveness. The incorporation of polymers, ligands or nanoparticles that have an affinity for the therapeutic of interest improve control over the release of small-molecule drugs and proteins from hydrogels, enabling spatial and temporal control over the delivery. Such affinity-based strategies can overcome drug burst release and challenges associated with protein instability, allowing more effective therapeutic molecule delivery for a range of applications from therapeutic contact lenses to ischemic tissue regeneration. PMID:27403513

  19. Purification and assay of secretory lithostathine in human pancreatic juice by fast protein liquid chromatography.

    PubMed Central

    Mariani, A; Mezzi, G; Malesci, A

    1995-01-01

    Impaired secretion of lithostathine, a pancreatic glycoprotein capable of inhibiting the growth of CaCO3 crystals, has been reported in chronic calcifying pancreatitis. Controversial results were obtained, however, using immunoassays with different antibodies. The aim of this study was to purify and to measure juice lithostathine by a non-immunological method. Fast protein liquid chromatography (FPLC) on a cation exchange column eluted by a sodium chloride gradient, was used. The conditions appropriate to separate secretory (S) from hydrolysed (H) isoforms of immunopurified lithostathine were also used for juice analysis. Pancreatic juice was collected by endoscopic cannulation of the major pancreatic duct, after secretin stimulation, from eight patients with chronic pancreatitis (CP) and from eight controls. In all samples, S-isoforms of lithostathine (ranging from 16 to 19 Mr at SDS-PAGE) were the only constituent of two of the 15 peaks in which FPLC resolved the pancreatic proteins. The nature of these two peaks was confirmed by their coelution with immunopurified S-lithostathine and by immunoblot analysis with polyclonal anti-lithostathine antibodies. The ratio between the area of S-lithostathine peaks and the total area of proteic eluates, was always lower in CP patients (5.3 micrograms/mg of protein, median value; 0.2-15.4, range) than in controls (35.2 micrograms/mg; 16.6-55.9). It is concluded that lithostathine can be purified and measured in pancreatic juice by FPLC. Our results with a nonimmunological assay confirm a reduced secretion of lithostathine in patients with CP. Images Figure 2 Figure 4 Figure 5 PMID:7737574

  20. Affinity Adsorbents Based on Carriers Activated by Epoxy-compounds

    NASA Astrophysics Data System (ADS)

    Klyashchitskii, B. A.; Kuznetsov, P. V.

    1984-10-01

    The review is devoted to the synthesis and applications of affinity adsorbents based on carriers activated by epoxy-compounds. The methods for the introduction of epoxy-groups into carriers of different chemical types are discussed and conditions for the immobilisation of three-dimensional spacers and low-molecular-weight and polymeric ligands on carriers containing epoxy-groups are considered. Data are presented on the properties and applications of adsorbents of this type in affinity chromatography. The bibliography includes 144 references.

  1. Confirmation of immuno-reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity-CIEF.

    PubMed

    Dullnig, Verena; Weiss, Richard; Amon, Sabine; Rizzi, Andreas; Stutz, Hanno

    2009-07-01

    Affinity-CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno-chemically. For this purpose mAbs of the IgG-type have been produced in-lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS-PAGE, MALDI-TOF-MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering pI ranges of 6.98-7.09 and 6.78-7.03 for clones 2 and 5.1 with major peaks at pI 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (pI 4.95) were incubated with 2.0 mumol/L mIgG, novel peaks were progressively induced in a pI range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All pI values were calculated using two pI marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno-chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag-antibody complexes and Ag variants coexisting in one sample. PMID:19621361

  2. Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

    PubMed Central

    Murphy, Patrick J. M.

    2014-01-01

    Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in

  3. Revealing novel telomere proteins using in vivo cross-linking, tandem affinity purification, and label-free quantitative LC-FTICR-MS.

    PubMed

    Nittis, Thalia; Guittat, Lionel; LeDuc, Richard D; Dao, Ben; Duxin, Julien P; Rohrs, Henry; Townsend, R Reid; Stewart, Sheila A

    2010-06-01

    Telomeres are DNA-protein structures that protect chromosome ends from the actions of the DNA repair machinery. When telomeric integrity is compromised, genomic instability ensues. Considerable effort has focused on identification of telomere-binding proteins and elucidation of their functions. To date, protein identification has relied on classical immunoprecipitation and mass spectrometric approaches, primarily under conditions that favor isolation of proteins with strong or long lived interactions that are present at sufficient quantities to visualize by SDS-PAGE. To facilitate identification of low abundance and transiently associated telomere-binding proteins, we developed a novel approach that combines in vivo protein-protein cross-linking, tandem affinity purification, and stringent sequential endoprotease digestion. Peptides were identified by label-free comparative nano-LC-FTICR-MS. Here, we expressed an epitope-tagged telomere-binding protein and utilized a modified chromatin immunoprecipitation approach to cross-link associated proteins. The resulting immunoprecipitant contained telomeric DNA, establishing that this approach captures bona fide telomere binding complexes. To identify proteins present in the immunocaptured complexes, samples were reduced, alkylated, and digested with sequential endoprotease treatment. The resulting peptides were purified using a microscale porous graphite stationary phase and analyzed using nano-LC-FTICR-MS. Proteins enriched in cells expressing HA-FLAG-TIN2 were identified by label-free quantitative analysis of the FTICR mass spectra from different samples and ion trap tandem mass spectrometry followed by database searching. We identified all of the proteins that constitute the telomeric shelterin complex, thus validating the robustness of this approach. We also identified 62 novel telomere-binding proteins. These results demonstrate that DNA-bound protein complexes, including those present at low molar ratios, can be

  4. Biotinylated hyaluronan: a versatile and highly sensitive probe capable of detecting nanogram levels of hyaluronan binding proteins (hyaladherins) on electroblots by a novel affinity detection procedure.

    PubMed

    Melrose, J; Numata, Y; Ghosh, P

    1996-01-01

    Hyaluronan influences cellular proliferation and migration in developing, regenerating and remodelling tissues and in tissues undergoing malignant tumour-cell invasion. The widespread occurrence of hyaluronan-binding proteins indicates that the recognition of hyaluronan is important to tissue organisation and the control of cellular behaviour. A number of extracellular matrix and cellular proteins, which have been termed the hyaladherins, have specific affinities for hyaluronan. These include cartilage link-protein, hyaluronectin, neurocan, versican and aggrecan, which all bind to HA within the extracellular matrix. Cellular receptors for hyaluronan such as CD44 and RHAMM (receptor for hyaluronate-mediated motility) have also been identified. In the present study biotinylated hyaluronan (bHA) was prepared by reacting adipic dihydrazide with a 170 kDa hyaluronan sample using the bifunctional reagent 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide. The resultant free amine moeity of the hydrazido-hyaluronan was then reacted with biotin succinimidyl ester (sulfo-NHS-biotin) to prepare the bHA. After 4-20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotting to nitrocellulose membranes, bHA and avidin alkaline phosphatase conjugate could be used in conjunction with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrates to specifically visualise with high sensitivity (> or = 2 ng), bovine nasal cartilage link-protein, aggrecan hyaluronan binding region, and human fibroblast hyaluronan receptors such as CD-44. Conventional Western blotting using specific monoclonal antibodies to these proteins was also used to confirm the identities of these proteins. PMID:8907541

  5. Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

    PubMed Central

    Kaplan, W.; Hüsler, P.; Klump, H.; Erhardt, J.; Sluis-Cremer, N.; Dirr, H.

    1997-01-01

    A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol. PMID:9041642

  6. Morphometric affinities of gigantopithecus.

    PubMed

    Gelvin, B R

    1980-11-01

    Multivariate analyses, supplemented by univariate statistical methods, of measurements from mandibular tooth crown dimensions and the mandible of Gigantopithecus blacki, G. bilaspurensis, Plio-Plelstocene hominids, Homo erectus, and seven Neogene ape species from the genera Proconsul, Sivapithecus, Ouranopithecus, and Dryopithecus were used to assess the morphometric affinities of Gigantopithecus. The results show that Gigantopithecus displays affinities to Ouranopithecus and to the hominids, particularly the Plio-Plelstocene hominids, rather than to the apes. Ouranopithecus demonstrated dental resemblances to G. bilaspurensis and the Plio-Pleistocene hominids but mandibular similarities to the apes. Results of analyses of tooth and mandibular shape indices, combined with multivariate distance and temporal relationships, suggest that Ouranopithecus is a more likely candidate for Gigantopithecus ancestry than is Silvapithecus indicus. Shape and allometric differences between G. bilaspurensis and the robust australopithecines weaken the argument for an ancestral-descendant relationship between these groups. The results support the hypothesis that Gigantopithecus is an extinct side branch of the Hominidae. PMID:7468790

  7. Gas Chromatography

    NASA Astrophysics Data System (ADS)

    Qian, Michael C.; Peterson, Devin G.; Reineccius, Gary A.

    The first publication on gas chromatography (GC) was in 1952 (1), while the first commercial instruments were manufactured in 1956. James and Martin (1) separated fatty acids by GC, collected the column effluent, and titrated the individual fatty acids for quantitation. GC has advanced greatly since that early work and is now considered to be a mature field that is approaching theoretical limitations.

  8. Ion Chromatography.

    ERIC Educational Resources Information Center

    Mulik, James D.; Sawicki, Eugene

    1979-01-01

    Accurate for the analysis of ions in solution, this form of analysis enables the analyst to directly assay many compounds that previously were difficult or impossible to analyze. The method is a combination of the methodologies of ion exchange, liquid chromatography, and conductimetric determination with eluant suppression. (Author/RE)

  9. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  10. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented. PMID:26830537

  11. Recent advances in affinity capillary electrophoresis for binding studies.

    PubMed

    Albishri, Hassan M; El Deeb, Sami; AlGarabli, Noura; AlAstal, Raghda; Alhazmi, Hassan A; Nachbar, Markus; El-Hady, Deia Abd; Wätzig, Hermann

    2014-01-01

    The present review covers recent advances and important applications of affinity capillary electrophoresis (ACE). It provides an overview about various ACE types, including ACE-MS, the multiple injection mode, the use of microchips and field-amplified sample injection-ACE. The most common scenarios of the studied affinity interactions are protein-drug, protein-metal ion, protein-protein, protein-DNA, protein-carbohydrate, carbohydrate-drug, peptide-peptide, DNA-drug and antigen-antibody. Approaches for the improvements of ACE in term of precision, rinsing protocols and sensitivity are discussed. The combined use of computer simulation programs to support data evaluation is presented. In conclusion, the performance of ACE is compared with other techniques such as equilibrium dialysis, parallel artificial membrane permeability assay, high-performance affinity chromatography as well as surface plasmon resonance, ultraviolet, circular dichroism, nuclear magnetic resonance, Fourier transform infrared, fluorescence, MS and isothermal titration calorimetry. PMID:25534793

  12. Affinity purification and mass spectrometry: an attractive choice to investigate protein-protein interactions in plant immunity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Affinity purification of protein complexes from biological tissues, followed by liquid chromatography- tandem mass spectrometry (AP-MS/MS), has ballooned in recent years due to sizeable increases in nucleic acid sequence data essential for interpreting mass spectra, improvements in affinity purifica...

  13. Minibodies and Multimodal Chromatography Methods

    PubMed Central

    Cheung, Chia-Wei; Lepin, Eric J.; Wu, Anna M.; Sherman, Mark A.; Raubitschek, Andrew A.; Yazaki, Paul J.

    2011-01-01

    This case study describes early phase purification process development for a recombinant anticancer minibody produced in mammalian cell culture. The minibody did not bind to protein A. Cation-exchange, anion-exchange, hydrophobic-interaction, and hydroxyapatite (eluted by phosphate gradient) chromatographic methods were scouted, but the minibody coeluted with BSA to a substantial degree on each. Hydroxyapatite eluted with a sodium chloride gradient separated BSA and also removed a dimeric contaminant, but BSA consumed so much binding capacity that this proved impractical as a capture tool. Capto MMC media proved capable of supporting adequate capture and significant dimer removal, although both loading and elution selectivity varied dramatically with the amount of supernatant applied to the column. An anion-exchange step was included to fortify overall virus and DNA removal. These results illustrate the value of multimodal chromatography methods when affinity chromatography methods are lacking and conventional alternatives prove inadequate. PMID:21984873

  14. Evaluation of Quantitative Performance of Sequential Immobilized Metal Affinity Chromatographic Enrichment for Phosphopeptides

    PubMed Central

    Sun, Zeyu; Hamilton, Karyn L.; Reardon, Kenneth F.

    2014-01-01

    We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium-dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. While our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semi-quantitative method when used in large-scale phosphoproteomics studies in complex backgrounds. PMID:24096195

  15. Inhibition of the hemorrhagic and proteolytic activities of Lansberg's hognose pit viper (Porthidium lansbergii hutmanni) venom by opossum (Didelphis marsupialis) serum: isolation of Didelphis marsupialis 0.15Dm fraction on DEAE-cellulose chromatography.

    PubMed

    Pineda, María E; Girón, María E; Estrella, Amalid; Sánchez, Elda E; Aguilar, Irma; Fernandez, Irma; Vargas, Alba M; Scannone, Héctor; Rodríguez-Acosta, Alexis

    2008-01-01

    Earlier studies have revealed the ability of sera from several mammals to neutralize the toxic effects of snake venom. The Venezuelan opossum (Didelphis marsupialis) is one that has been found to inhibit hemorrhagic and proteolytic activities of venoms from many species of snakes. In this article it is shown that the opossum sera and its 0.15DM fraction were able to completely neutralize both hemorrhagic and hydrolysis (proteolysis) of casein effects induced by venom of the Lansberg's hognose pit viper (Porthidium lansbergii hutmanni). We have used DEAE-cellulose ion exchange chromatography to collect protein fractions from D. marsupialis sera which were able to defend mice from the lethal effects of P.l. hutmanni venom. The fractions separated were homogeneous by conventional electrophoresis using SDS-PAGE. The protein bands obtained contained molecular weights of approximately 6 to 220 kDa. These results revealed the presence of proteases inhibitors in the opossum sera fractions and the inhibition of venom activity by opossum sera suggesting a reciprocal adaptation at the molecular level. PMID:18800269

  16. Gas Chromatography

    NASA Astrophysics Data System (ADS)

    Qian, Michael C.

    Gas chromatography (GC) has many applications in the analysis of food products. GC has been used for the determination of fatty acids, triglycerides, cholesterol, gases, water, alcohols, pesticides, flavor compounds, and many more. While GC has been used for other food components such as sugars, oligosaccharides, amino acids, peptides, and vitamins, these substances are more suited to analysis by high performance liquid chromatography. GC is ideally suited to the analysis of volatile substances that are thermally stable. Substances such as pesticides and flavor compounds that meet these criteria can be isolated from a food and directly injected into the GC. For compounds that are thermally unstable, too low in volatility, or yield poor chromatographic separation due to polarity, a derivatization step must be done before GC analysis. The two parts of the experiment described here include the analysis of alcohols that requires no derivatization step, and the analysis of fatty acids which requires derivatization. The experiments specify the use of capillary columns, but the first experiment includes conditions for a packed column.

  17. The crystal structure of oxy hemoglobin from high oxygen affinity bird emu (Dromaius novaehollandiae).

    PubMed

    Mohamed Abubakkar, Mohamed H; Saraboji, Kadhirvel; Ponnuswamy, Mon Nanjappa G

    2014-01-01

    Hemoglobin is an honorary enzyme, a two-way respiratory carrier, transporting oxygen from the lungs to the tissues and facilitating the return transport of carbon dioxide. Hemoglobin has high affinity for oxygen and low affinity for carbon dioxide and other substances in the arterial circulation, whereas in the venous circulation these relative affinities are upturned. The oxygen affinity of hemoglobin increases with the fall in temperature and decreases with the increase in pH and 2, 3-bisphosphoglycerate; point mutations also affect the tetrameric arrangement and alter the oxygen affinity. Though several studies have revealed the specific reasons for the adaptation of increased oxygen affinity of avian hemoglobins at high-altitudes, further structural insights on hemoglobins from high oxygen affinity species are required to understand the detailed oxygen adaptation at the molecular level. Herein, we describe the structural investigation of hemoglobin from emu (Dromaius novaehollandiae), a high oxygen affinity bird. Hemoglobin from emu was purified using anion-exchange chromatography, crystallized and determined the structure in the oxy form at a resolution of 2.3 Å; the R-factor of the model was 19.2%. The structure was compared with other oxy hemoglobins of high oxygen affinity avian species; significant changes are noted at intra-subunit contacts which provide the clues for increased oxygen affinity of emu hemoglobin. PMID:25146185

  18. Affinity chromatography of yeast alpha-glucosidase using ligand-mediated chromatography on immobilized phenylboronic acids.

    PubMed Central

    Myöhänen, T A; Bouriotis, V; Dean, P D

    1981-01-01

    The synthesis of 3-nitro-4-(6-aminohexylamido)phenylboronic acid is described. The properties of two novel forms of immobilized phenylboronate agarose adsorbents [m-aminophenylboronic acid-Matrex Gel and 3-nitro-4-(6-aminohexylamido)phenylboronic acid-Sepharose CL-6B] were investigated. Both gels bind and selectively retard the glycoprotein alpha-glucosidase from yeast. The retardation is affected by following parameters: (i) pH, (ii) presence of sugar, (iii) concentration of sugar and (iv) buffer species (especially triethanolamine). Five sugars were studied, namely sorbitol, fructose, ribose, glucose and maltose. The concentration of sugar required to produce significant retardation increased in the above order, whereas the ability of sugar to form a complex with boron decreases in the same order. These effects were observed with crude as well as pure enzyme. Since alpha-glucosidase is a glycoprotein, it is proposed that this protein is mainly bound to these immobilized phenylboronates via sugar (glyco) residues. Displacement of the enzyme from the column is effected by the sugar in the buffer (or in a preincubation mixture). However, the marked pH-dependence (this retardation effect could only be observed at pH 7.4) suggests that these results are not due solely to hydrophobic or ionic mechanisms and are more complex than simple sugar-phenylboronic acid interactions. PMID:7034722

  19. Instrumentation: Ion Chromatography.

    ERIC Educational Resources Information Center

    Fritz, James S.

    1987-01-01

    Discusses the importance of ion chromatography in separating and measuring anions. The principles of ion exchange are presented, along with some applications of ion chromatography in industry. Ion chromatography systems are described, as well as ion pair and ion exclusion chromatography, column packings, detectors, and programming. (TW)

  20. Purification and properties of chicken egg-white cobalamin-binding protein.

    PubMed

    Zaman, K; Zak, Z

    1990-10-01

    A cobalamin-binding protein has been purified from chicken egg-white by using a combination of conventional and high performance ion-exchange chromatography. Following initial purification by DEAE-cellulose, ammonium sulphate precipitation, Sephacryl S-200 CM-cellulose and affinity chromatography, appropriate fractions were further purified using the Pharmacia fast protein liquid chromatography (FPLC) system. Using this method of purification, egg-white CBP has been purified more rapidly and with greater recovery than with conventional column chromatography. The homogeneity of this protein was verified by SDS-PAGE. The Mr was 37,000 by SDS-PAGE and 39,000 by gel filtration, which indicated that it was a glycoprotein. The stokes radius was 4.1 nm and pI was 4.3. The protein bound 57COB12 with a molar ratio of 1/1 and kd of 0.40 microM. The egg-white CBP was composed of 294 amino acid residues. Thiol groups and metal ions were not connected with the Cbl-binding activities. PMID:2078588

  1. Electrochemical affinity biosensors for detection of mycotoxins: A review.

    PubMed

    Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

    2013-11-15

    This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago. PMID:23743326

  2. Stable high capacity, F-actin affinity column

    SciTech Connect

    Luna, E.J.; Wang, Y.L.; Voss, E.W. Jr.; Branton, D.; Taylor, D.L.

    1982-11-10

    A high capacity F-actin affinity matrix is constructed by binding fluorescyl-actin to rabbit anti-fluorescein IgG that is covalently bound to Sepharose 4B. When stabilized with phalloidin, the actin remains associated with the Sepharose beads during repeated washes, activates the ATPase activity of myosin subfragment 1, and specifically binds /sup 125/I-heavy meromyosin and /sup 125/I-tropomyosin. The associations between the F-actin-binding proteins are monitored both by affinity chromatography and by a rapid, low speed sedimentation assay. Anti-fluorescein IgG-Sepharose should be generally useful as a matrix for the immobilization of proteins containing accessible, covalently bound fluorescein groups.

  3. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Sondek, John

    2004-09-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, and for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:18429272

  4. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Brill, Allison L; Pasker, Renee L

    2013-01-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:24510596

  5. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  6. Quantifying Affinity among Chinese Dialects.

    ERIC Educational Resources Information Center

    Cheng, Chin-Chuan

    A study of the relationships between Chinese dialects based on a quantitative measure of dialect affinity is summarized. First, tone values in all the dialect localities available in the early 1970s were used to calculate the dialectal differences in terms of tone height with respect to the "yin and yang" split. In the late 1970s, calculations of…

  7. Identification of pregnancy-associated glycoproteins by peptide mass fingerprinting in water buffalo (Bubalus bubalis).

    PubMed

    Kumar, Pradeep; Saxena, Abhishake; Singh, S K; Sharma, R K; Singh, I; Agarwal, S K

    2014-08-01

    Ruminant placentas synthesize pregnancy-associated glycoproteins (PAGs) during pregnancy, which serve as biomarkers of pregnancy. The present study was conducted to verify, whether PAGs are expressed in buffalo placenta by using lectin-based affinity chromatography and peptide mass finger printing (PMF). Fetal cotyledonary tissues were collected from gravid uteri procured from slaughtered house. Proteins were extracted and subjected to wheat germ agglutinin (WGA) lectin affinity chromatography to isolate the PAGs. The isolated glycoproteins were separated by one-dimensional SDS-PAGE. PMF results of the 75 kDa protein revealed presence of two PAGs (PAG-7 and -11). The PAG-7 consisted of about 170 mass signals, of which 16 were assigned to corresponding/translated cDNA sequences of buffalo PAG-7, leading to sequence coverage of 40%. PMF result of PAG-11 showed 170 mass signals, of which 15 were assigned to buffalo PAG-11, leading to sequence coverage of 34%. In conclusion, the glycoprotein isolated from placental extract corresponding to 75 kDa band on SDS PAGE gel was a mixture of PAG-7 and -11, which may help in development of suitable diagnostics for pregnancy in buffalo. PMID:25296505

  8. Development and application of high-performance affinity beads: toward chemical biology and drug discovery.

    PubMed

    Sakamoto, Satoshi; Kabe, Yasuaki; Hatakeyama, Mamoru; Yamaguchi, Yuki; Handa, Hiroshi

    2009-01-01

    In drug development research, the elucidation and understanding of the interactions between physiologically active substances and proteins that numerous genes produce is important. Currently, most commercially available drugs and physiologically active substances have been brought to market without knowledge of factors interacting with the drugs and the substances. Affinity purification is a useful and powerful technique employed to understand factors that are targeted by drugs and physiologically active substances. However, use of conventional matrices for affinity chromatography often causes a decrease in efficiency of affinity purification and, as a result, more practical matrices for affinity purification have been developed for application in drug discovery research. In this paper, we describe the development of high-performance affinity beads (SG beads and FG beads) that enable one-step affinity purification of drug targets and the elucidation of the mechanism of the action of the drugs. We also describe a chemical screening system using our affinity beads. We hope that utilization of the affinity beads will contribute to the progress of research in chemical biology. PMID:19243077

  9. Application of high-speed counter-current chromatography coupled with a reverse micelle solvent system to separate three proteins from Momordica charantia.

    PubMed

    Li, Yingnan; Yin, Lianhong; Zheng, Lingli; Xu, Lina; Xu, Youwei; Zhao, Yanyan; Qi, Yan; Yao, Jihong; Han, Xu; Liu, Kexin; Peng, Jinyong

    2012-05-01

    High-speed counter-current chromatography (HSCCC) coupled with a reverse micelle solvent system was successfully developed to separate three proteins from Momordica charantia. Suitable HSCCC conditions were carefully optimized as follows: the stationary phase was a reverse micellar phase composed of isooctane and 50mM bis-(2-ethylhexyl)-1-sulfosuccinate sodium (AOT). The mobile phase contained mobile phase A (50mM Tris-HCl buffer containing 50mM KCl at pH 7.0) for forward-extraction and mobile phase B (50mM Tris-HCl buffer containing 0.5M KCl at pH 10.0) for back-extraction. The flow rate, detection wavelength and column temperature were set at 1.5 ml/min, 280 nm and 4 °C, respectively. Under these conditions, three fractions (I, II and III) were separated, which showed high purity when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The structures of these proteins were then identified by MALDI-TOF/TOF-MS/MS and compared with the NCBInr database. Fractions I and III were identified as resistance-like protein P-B and pentatricopeptide repeat-containing protein, respectively, which were found in M. charantia for the first time. However, fraction II, which is thought to be a new protein, was not identified, and further investigations on this fraction are required. The anticancer activities of these three proteins on the human gastric cancer cell line SGC-7901 were evaluated in vitro. The results indicated that fraction II has excellent anticancer activity (IC(50)=0.116 mg/ml for 48 h treatment). This is the first report on the use of HSCCC to isolate proteins from M. charantia. PMID:22465200

  10. Biochemical characterization of hemoglobins from Caspian Sea sturgeons (Acipenser persicus and Acipenser stellatus).

    PubMed

    Ariaeenejad, Shohreh; Habibi-Rezaei, Mehran; Jamili, Shahla; Fatemi, Mohammad Reza; Poursasan, Najmeh; Ahmad, Faizan; Sheibani, Nader; Kavousi, Kaveh; Moosavi-Movahedi, Ali A

    2012-01-01

    Hemoglobin (Hb) variability is a commonly used index of phylogenetic differentiation and molecular adaptation in fish enabling them to adapt to different ecological conditions. In this study, the characteristics of Hbs from two Sturgeon species of the Southern Caspian Sea Basin were investigated. After extraction and separation of hemoglobin from whole blood, the polyacrylamide gel electrophoresis (SDS-PAGE), cellulose acetate electrophoresis, and isoelectric focusing (IEF) were used to confirm Hb variabilities in these fishes. We showed that although both species have variable Hbs with different isoelectric points, their dominant Hbs can be identified. Ion exchange on CM-cellulose chromatography was used for purification of the dominant Hbs from these fishes. The accuracy of the methods was confirmed by IEF and SDS-PAGE. Spectral studies using fluorescence spectrophotometery indicated that although the Hbs from these fishes had similar properties they exhibited clear differences with human Hb. A comparative study of Hbs alpha-helix secondary substructures was performed by circular dichroism spectropolarimetry (CD) analysis. UV-vis spectrophotometery was also utilized to measure oxygen affinity of Hbs by sodium dithionite. Oxygen affinities of these Hbs were compared using Hb-oxygen dissociation curves. Together, these results demonstrate a significant relationship between oxygen affinity of fish hemoglobins and environmental partial pressure of oxygen. PMID:21833671

  11. Theoretical proton affinity and fluoride affinity of nerve agent VX.

    PubMed

    Bera, Narayan C; Maeda, Satoshi; Morokuma, Keiji; Viggiano, Al A

    2010-12-23

    Proton affinity and fluoride affinity of nerve agent VX at all of its possible sites were calculated at the RI-MP2/cc-pVTZ//B3LYP/6-31G* and RI-MP2/aug-cc-pVTZ//B3LYP/6-31+G* levels, respectively. The protonation leads to various unique structures, with H(+) attached to oxygen, nitrogen, and sulfur atoms; among which the nitrogen site possesses the highest proton affinity of -ΔE ∼ 251 kcal/mol, suggesting that this is likely to be the major product. In addition some H(2), CH(4) dissociation as well as destruction channels have been found, among which the CH(4) + [Et-O-P(═O)(Me)-S-(CH(2))(2)-N(+)(iPr)═CHMe] product and the destruction product forming Et-O-P(═O)(Me)-SMe + CH(2)═N(+)(iPr)(2) are only 9 kcal/mol less stable than the most stable N-protonated product. For fluoridization, the S-P destruction channel to give Et-O-P(═O)(Me)(F) + [S-(CH(2))(2)-N-(iPr)(2)](-) is energetically the most favorable, with a fluoride affinity of -ΔE ∼ 44 kcal. Various F(-) ion-molecule complexes are also found, with the one having F(-) interacting with two hydrogen atoms in different alkyl groups to be only 9 kcal/mol higher than the above destruction product. These results suggest VX behaves quite differently from surrogate systems. PMID:21117653

  12. Chromatography: concepts and contrasts

    SciTech Connect

    Miller, J.M.

    1988-01-01

    As the author states in the Preface, this text attempts to provide a unified approach to chromatography (hence the title) by way of contrasting similarities and differences between gas chromatography (GC), column liquid chromatography (LC), and thin-layer chromatography (TLC). This book is also said to be pitched at an elementary level, suitable for most newcomers to the field (e.g., advanced undergraduates and beginning graduate students in the academic world, as well as bench-level chemists in industry).

  13. Galactinol synthase from kidney bean cotyledon and zucchini leaf. Purification and N-terminal sequences.

    PubMed Central

    Liu, J J; Odegard, W; de Lumen, B O

    1995-01-01

    Galactinol synthase (GS) was purified 1591-fold with a 3.9% recovery from the cotyledon of kidney bean (Phaseolus vulgaris) by a novel scheme consisting of ammonium sulfate fractionation followed by diethylaminoethyl, Affi-Gel Blue, and UDP-hexanolamine affinity chromatography. The purified enzyme had a specific activity of 8.75 mumol mg-1 min-1, a pH optimum of 7.0, and requirements for manganese ion and DTT. The enzyme exhibited a Km = 0.4 mM for UDP-galactose and a Km = 4.5 mM for myo-inositol. It was identified as a 38-kD peptide that co-purified with a 41- and a 43-kD peptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purification to homogeneity was achieved by isolating the 38-kD peptide from the SDS-PAGE gel. To clarify conflicting reports in the literature about the relative molecular mass of purified GS from zucchini leaf (Cucurbita pepo), a similar scheme with modified eluting conditions was used to purify GS from this source. Zucchini leaf GS was purified to homogeneity and identified as a 36-kD peptide on SDS-PAGE. Partial N-terminal sequences of the 38-kD peptide from kidney bean cotyledon and the 36-kD peptide from zucchini leaf were obtained. To facilitate identification of GS during the purification, an assay utilizing thin-layer chromatography and an isotopic analytic imaging scanner was developed. PMID:7480343

  14. Polymer displacement/shielding in protein chromatography.

    PubMed

    Kumar, A; Galaev, I Y; Mattiasson, B

    2000-05-12

    An overview of different applications of polymer interactions with ion-exchange and dye-affinity chromatographic matrices is presented here. The strength of interaction between the ligand and the polymer plays a crucial role in deciding the mode of chromatographic application. Charged, non-ionic and thermosensitive polymers such as poly(ethylene imine), poly(N-vinyl pyrrolidone) and poly(vinyl caprolactam) respectively, show different degrees of interaction with the dye molecules in dye ligand chromatography. Polymers, with their ability of multipoint and hence strong attachment to the chromatographic matrices, were used as efficient displacers in displacement chromatography. The polymer displacement resulted in better recoveries and sharper elution profiles than traditional salt elutions. The globule-coil transition of the thermosensitive reversible soluble-insoluble polymer, poly(vinyl caprolactam), can be exploited in dye-affinity columns for the temperature induced displacement of the bound protein. In another situation, prior to the column chromatography of crude protein extract, polymers formed complexes with the dye matrix and "shielded" the column. The polymer shielding decreased the nonspecific interactions without affecting the specific interactions of the target protein to the dye matrix. PMID:10872581

  15. Immunochemical studies of the muscarinic acetylcholine receptor.

    PubMed

    André, C; Marullo, S; Guillet, J G; Convents, A; Lauwereys, M; Kaveri, S; Hoebeke, J; Strosberg, A D

    1987-01-01

    Muscarinic receptors have been purified from calf forebrain plasma cell membranes by affinity chromatography on a dexetimide-agarose gel. SDS-PAGE analysis showed a single 70 kDa band. Monoclonal antibodies have been prepared against these affinity purified 70 kDa protein(s). One antibody, M-35, immunoprecipitated up to 80% of digitonin-solubilized muscarinic receptors. M-35 had agonist-like effects on guinea-pig myometrium: it increased the intracellular cyclic GMP content, decreased prostaglandin-induced cyclic AMP accumulation and caused muscle contractions. The two first effects were inhibited by atropine. M-35 was used to visualize muscarinic receptors at the surface of human fibroblastic cells. In the particular cell line used, the receptors have a low affinity for pirenzepine, were negatively coupled to adenylate cyclase and mediated increase in the phosphatidyl-inositol breakdown. PMID:3040987

  16. Crystal structures of fusion proteins with large-affinity tags.

    PubMed

    Smyth, Douglas R; Mrozkiewicz, Marek K; McGrath, William J; Listwan, Pawel; Kobe, Bostjan

    2003-07-01

    The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest. PMID:12824478

  17. Improved Thermal Modulator for Gas Chromatography

    NASA Technical Reports Server (NTRS)

    Hasselbrink, Ernest Frederick, Jr.; Hunt, Patrick J.; Sacks, Richard D.

    2008-01-01

    An improved thermal modulator has been invented for use in a variant of gas chromatography (GC). The variant in question denoted as two-dimensional gas chromatography (2DGC) or GC-GC involves the use of three series-connected chromatographic columns, in the form of capillary tubes coated interiorly with suitable stationary phases (compounds for which different analytes exhibit different degrees of affinity). The two end columns are relatively long and are used as standard GC columns. The thermal modulator includes the middle column, which is relatively short and is not used as a standard GC column: instead, its temperature is modulated to affect timed adsorption and desorption of analyte gases between the two end columns in accordance with a 2DGC protocol.

  18. [Purification of lectin from perch (Persa fluviatilis L.) roe specific to cellobiose and study of its characteristics].

    PubMed

    Antoniuk, V O

    2004-01-01

    Two lectins with different carbohydrate specificity were purified from perch (Persa fluviatilis L.) roe (coastal ecological form) by affinity chromatography on ovariomucine H-sepharose from a human ovary cyst. One lectin was eluted by cellobiose and another lectin was eluted by L-fucose. The L-fucose-specific lectin interacted only with L-fucose and its derivatives, but did not interact with cellobiose and salicin. The cellobiose-specific lectin interacted with all the examined carbohydrates, but cellobiose was the best inhibitor. This lectin can be also purified on cellulose as an affinity sorbent. Unlike the L-fucose-specific lectin from perch roe, the cellobiose-specific lectin is less soluble in water-saline solutions. Lectin solubility increases greatly in presence of specific inhibitors, cellobiose, in particular. L-fucose, alpha-methyl-L-fucopyranoside and 4-nitrophenyl-alpha-L-fucopyranoside are equivalent inhibitors for both lectins. According to SDS-PAGE data, the lectins contain two components with molecular weight 12-13 kDa. In solutions, these components form molecules with 50 or 100 kDa (depending on pH). Data obtained from electrophoresis in PAAG in alkaline (pH 8.9) and acidic system (pH 4.3), and SDS-PAGE did not display essential distinctions between these both lectins. PMID:15909420

  19. Purification of PRL receptors from toad kidney: Comparisons with rabbit mammary PRL receptors

    SciTech Connect

    Dunand, M.; Kraehenbuhl, J.P.; Rossier, B.C.; Aubert, M.L. Univ. of Lausanne School of Medicine )

    1988-03-01

    The binding characteristics of the prolactin (PRL) receptors present in toad (Bufo marinus) kidneys were investigated and compared to those of PRL receptors present in rabbit mammary glands. The molecular characteristics of the Triton X-100 solubilized renal and mammary PRL receptors were assessed by gel filtration and by migration analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after affinity labeling of the binding sites with {sup 125}I-human growth hormone. Similar results were obtained for both receptors. Partial purification of the toad PRL receptor could be achieved by affinity chromatography. The molecular weight of this purified receptor could be determined by analysis of SDS-PAGE. With the use of a polyclonal antiserum raised against a purified preparation of rabbit mammary PRL receptor, one or several antigenic epitope(s) could be identified on the core of the toad renal PRL receptor. In conclusion, although the structure and the biological role(s) of PRL have substantially changed during evolution, the receptor for this hormone has retained many of its structural features as could be assessed between an amphibian and a mammalian species on functionally different target tissues.

  20. Indian craniometric variability and affinities.

    PubMed

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with "Caucasoid" populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

  1. Indian Craniometric Variability and Affinities

    PubMed Central

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with “Caucasoid” populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

  2. A novel affinity disks for bovine serum albumin purification.

    PubMed

    Tuzmen, Nalan; Kalburcu, Tülden; Uygun, Deniz Aktaş; Akgol, Sinan; Denizli, Adil

    2015-01-01

    The adsorption characteristics of bovine serum albumin (BSA) onto the supermacroporous poly(hydroxyethylmethacrylate)-Reactive Green 19 [p(HEMA)-RG] cryogel disks have been investigated in this paper. p(HEMA) cryogel disks were prepared by radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Reactive Green (RG) 19 was covalently attached to the p(HEMA) cryogel disks. These disks were used in BSA adsorption studies to interrogate the effects of pH, initial protein concentration, ionic strength, and temperature. BSA adsorption capacity of the p(HEMA)-RG cryogel disk was significantly improved after the incorporation of RG. Adsorption capacity reached a plateau value at about 0.8 mg/mL at pH 4.0. The amount of adsorbed BSA decreased from 37.7 to 13.9 mg/g with increasing NaCl concentration. The enthalpy of BSA adsorption onto the p(HEMA)-RG cryogel disk was calculated as -58.4 kJ/mol. The adsorption equilibrium isotherm was fitted well by the Freundlich model. BSA was desorbed from cryogel disks (over 90 %) using 0.5 M NaSCN, and the purity of desorbed BSA was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The experimental results showed that the p(HEMA)-RG cryogel disks have potential for the quick protein separation and purification process. PMID:25308615

  3. Purification, characterization and cloning of a chymotrypsin inhibitor (CI-9) from the hemolymph of the silkworm, Bombyx mori.

    PubMed

    Zhao, Ping; Xia, Qingyou; Li, Juan; Fujii, Hiroshi; Banno, Yutaka; Xiang, Zhonghuai

    2007-08-01

    Hemolymph chymotrypsin inhibitor 9 (CI-9) from the hemolymph of the silkworm, Bombyx mori, was purified by ammonium sulfate precipitation, Butyl Toyopearl hydrophobic chromatography, gel filtration through Sephadex C-50 and chymotrypsin-sepharose 4B affinity chromatography. Checked by Native PAGE and SDS-PAGE in combination with silver staining, the final preparation appeared homogeneous. In tricine SDS-PAGE, CI-9 displayed a molecular weight of 7.5 kD, which was determined to be 7167 Da with the Voyager TOFMass analyser. The pI value for CI-9, revealed by 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis), was 4.3. CI-9 exhibited inhibitory activity at a temperature as high as 100 degrees C and a stability against a wide range of pH (1-12). In N-terminal amino-acid analysis of CI-9, 40 amino acid residues were obtained. The C-terminal 22 amino acid residues were deduced by subsequently cloned cDNA and genomic fragments. MW and pI of CI-9 were predicted to be 7170.98 Da and 4.61, respectively, on the website. Its low molecular weight, high stability, conserved active site and Kunitz domain showed that CI-9 is a Kunitz-type CI. The difference of sequence and pI between CI-9 and other Kunitz type CIs indicated that it is a novel chymotrypsin inhibitor. PMID:17503165

  4. Hevea brasiliensis cell suspension peroxidase: purification, characterization and application for dye decolorization

    PubMed Central

    2013-01-01

    Peroxidases are oxidoreductase enzymes produced by most organisms. In this study, a peroxidase was purified from Hevea brasiliensis cell suspension by using anion exchange chromatography (DEAE-Sepharose), affinity chromatography (Con A-agarose) and preparative SDS-PAGE. The obtained enzyme appeared as a single band on SDS-PAGE with molecular mass of 70 kDa. Surprisingly, this purified peroxidase also had polyphenol oxidase activity. However, the biochemical characteristics were only studied in term of peroxidase because similar experiments in term of polyphenol oxidase have been reported in our pervious publication. The optimal pH of the purified peroxidase was 5.0 and its activity was retained at pH values between 5.0–10.0. The enzyme was heat stable over a wide range of temperatures (0–60°C), and less than 50% of its activity was lost at 70°C after incubation for 30 min. The enzyme was completely inhibited by β-mercaptoethanol and strongly inhibited by NaN3; in addition, its properties indicated that it was a heme containing glycoprotein. This peroxidase could decolorize many dyes; aniline blue, bromocresol purple, brilliant green, crystal violet, fuchsin, malachite green, methyl green, methyl violet and water blue. The stability against high temperature and extreme pH supported that the enzyme could be a potential peroxidase source for special industrial applications. PMID:23402438

  5. Purification and characterization of Locusta migratoria chymotrypsin.

    PubMed

    Sakal, E; Applebaum, S W; Birk, Y

    1988-12-01

    A chymotrypsin-like enzyme (CTLE) was isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides by ion-exchange chromatography on diethylaminoethyl (DEAE) cellulose followed by affinity chromatography on phenylbutylamine (PBA) Sepharose. The purity and homogeneity of CTLE have been shown by SDS-PAGE and on cellulose acetate strips. The enzyme has a molecular weight of 24,000, determined by SDS-PAGE and on a Sephadex G-75 calibrated column. It has an isoelectric point of 10.1 and contains 0-1 half cystine residues. Sequence analysis of the first 20 N-terminal amino acids has shown 25% homology with bovine chymotrypsin and 40% homology with Vespa crabo and Vespa orientalis chymotrypsins and with Hypoderma lineatum trypsin. The optimal pH for enzyme activity and stability was in the range of 8.5-9.0. The Km and kcat values, determined on substrates for proteolytic, esterolytic and amidolytic activity, similar to those for bovine chymotrypsin. CTLE was inactivated by PMSF and TPCK indicating the involvement of serine and histidine in its active site. The enzyme was fully inhibited by the proteinaceous, double-headed, chymotrypsin-trypsin inhibitors BBI from soybeans and CI from chickpeas, by chicken ovomucoid (COM) and turkey ovomucoid (TOM), as well as by the Kunitz soybean trypsin inhibitor (STI) which hardly inhibits bovine chymotrypsin. Inhibition studies of CTLE with amino acid and peptide-chloromethylketones point towards the existence of an extended binding site. PMID:3246483

  6. Identification of Tetraspanin-7 as a Target of Autoantibodies in Type 1 Diabetes.

    PubMed

    McLaughlin, Kerry A; Richardson, Carolyn C; Ravishankar, Aarthi; Brigatti, Cristina; Liberati, Daniela; Lampasona, Vito; Piemonti, Lorenzo; Morgan, Diana; Feltbower, Richard G; Christie, Michael R

    2016-06-01

    The presence of autoantibodies to multiple-islet autoantigens confers high risk for the development of type 1 diabetes. Four major autoantigens are established (insulin, glutamate decarboxylase, IA2, and zinc transporter-8), but the molecular identity of a fifth, a 38-kDa membrane glycoprotein (Glima), is unknown. Glima antibodies have been detectable only by immunoprecipitation from extracts of radiolabeled islet or neuronal cells. We sought to identify Glima to enable efficient assay of these autoantibodies. Mouse brain and lung were shown to express Glima. Membrane glycoproteins from extracts of these organs were enriched by detergent phase separation, lectin affinity chromatography, and SDS-PAGE. Proteins were also immunoaffinity purified from brain extracts using autoantibodies from the sera of patients with diabetes before SDS-PAGE. Eluates from gel regions equivalent to 38 kDa were analyzed by liquid chromatography-tandem mass spectrometry for protein identification. Three proteins were detected in samples from the brain and lung extracts, and in the immunoaffinity-purified sample, but not in the negative control. Only tetraspanin-7, a multipass transmembrane glycoprotein with neuroendocrine expression, had physical characteristics expected of Glima. Tetraspanin-7 was confirmed as an autoantigen by demonstrating binding to autoantibodies in type 1 diabetes. We identify tetraspanin-7 as a target of autoimmunity in diabetes, allowing its exploitation for diabetes prediction and immunotherapy. PMID:26953162

  7. Purification, cDNA cloning and functional expression of NADPH-cytochrome P450 reductase from Centaurium erythraea cell cultures.

    PubMed

    Schwarz, H; Liu, B; Peters, S; Barillas, W; Beerhues, L

    2009-05-01

    Solubilised NADPH-cytochrome P450 reductase (CPR) was purified from the microsomal fraction of centaury (Centaurium erythraea) cell cultures by Q-anion exchange chromatography and affinity chromatography on adenosine 2',5'-diphosphate agarose. SDS-PAGE demonstrated the presence of three CPR isoforms with molecular masses of 77, 79 and 81 kDa. The 79- and 81-kDa isoforms were identified as glycoproteins when blotted following SDS-PAGE and subjected to a sugar detection procedure. A homology-based approach led to the isolation of a CPR cDNA encoding the 77-kDa isoform. The enzyme was a class I CPR, possessing a short N-terminus upstream of the membrane anchor. The amino acid sequence contained a putative N-glycosylation site, indicating that the two major isoforms of 77 and 79 kDa are related through attachment of an oligosaccharide chain. This glycosylation process was also found upon heterologous expression in yeast. When co-expressed in yeast together with centaury coniferyl alcohol 5-hydroxylase, CPR efficiently supported the activity of the P450 enzyme. The genome of C. erythraea was found to contain a second CPR gene. RT-PCR experiments using gene-specific primers revealed differential regulation of the two CPR genes. While CPR 2 mRNA was strongly induced by the addition of methyl jasmonate to the cell cultures, the CPR 1 expression level did not change after this elicitation. PMID:19470102

  8. Chromatography resin support

    DOEpatents

    Dobos, James G.

    2002-01-01

    An apparatus and method of using an improved chromatography resin support is disclosed. The chromatography support platform is provided by a stainless steel hollow cylinder adapted for being inserted into a chromatography column. An exterior wall of the stainless steel cylinder defines a groove for carrying therein an "O"-ring. The upper surface of the stainless steel column is covered by a fine stainless steel mesh welded to the edges of the stainless steel cylinder. When placed upon a receiving ledge defined within a chromatography column, the "O"-ring provides a fluid tight seal with the inner edge wall of the chromatography cylinder. The stainless steel mesh supports the chromatography matrix and provides a back flushable support which is economical and simple to construct.

  9. Fun with Paper Chromatography.

    ERIC Educational Resources Information Center

    Coleman, Dava; Hounshell, Paul B.

    1982-01-01

    Discusses paper chromatographic techniques and provides examples of typical classroom activities. Includes description of retardation values obtained during chromatography exercises and suggests using them for math lessons. (JN)

  10. Affine hypersurfaces with parallel difference tensor relative to affine α-connection

    NASA Astrophysics Data System (ADS)

    Li, Cece

    2014-12-01

    Li and Zhang (2014) studied affine hypersurfaces of R n + 1 with parallel difference tensor relative to the affine α-connection ∇ (α), and characterized the generalized Cayley hypersurfaces by K n - 1 ≠ 0 and ∇ (α) K = 0 for some nonzero constant α, where the affine α-connection ∇ (α) of information geometry was introduced on affine hypersurface. In this paper, by a slightly different method we continue to study affine hypersurfaces with ∇ (α) K = 0, if α = 0 we further assume that the Pick invariant vanishes and affine metric is of constant sectional curvature. It is proved that they are either hyperquadrics or improper affine hypersphere with flat indefinite affine metric, the latter can be locally given as a graph of a polynomial of at most degree n + 1 with constant Hessian determinant. In particular, if the affine metric is definite, Lorentzian, or its negative index is 2, we complete the classification of such hypersurfaces.

  11. The maximal affinity of ligands

    PubMed Central

    Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A.

    1999-01-01

    We explore the question of what are the best ligands for macromolecular targets. A survey of experimental data on a large number of the strongest-binding ligands indicates that the free energy of binding increases with the number of nonhydrogen atoms with an initial slope of ≈−1.5 kcal/mol (1 cal = 4.18 J) per atom. For ligands that contain more than 15 nonhydrogen atoms, the free energy of binding increases very little with relative molecular mass. This nonlinearity is largely ascribed to nonthermodynamic factors. An analysis of the dominant interactions suggests that van der Waals interactions and hydrophobic effects provide a reasonable basis for understanding binding affinities across the entire set of ligands. Interesting outliers that bind unusually strongly on a per atom basis include metal ions, covalently attached ligands, and a few well known complexes such as biotin–avidin. PMID:10468550

  12. A comparative study of lectin affinity based plant n-glycoproteome profiling using tomato fruit as a model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lectin affinity chromatography (LAC) can provide a valuable front-end enrichment strategy for the study of N-glycoproteins and has been used to characterize a broad range eukaryotic N-glycoproteomes. Moreover, studies with mammalian systems have suggested that the use of multiple lectins with differ...

  13. Petasis-Ugi ligands: New affinity tools for the enrichment of phosphorylated peptides.

    PubMed

    Batalha, Íris L; Roque, Ana C A

    2016-09-15

    Affinity chromatography is a widespread technique for the enrichment and isolation of biologics, which relies on the selective and reversible interaction between affinity ligands and target molecules. Small synthetic affinity ligands are valuable alternatives due to their robustness, low cost and fast ligand development. This work reports, for the first time, the use of a sequential Petasis-Ugi multicomponent reaction to generate rationally designed solid-phase combinatorial libraries of small synthetic ligands, which can be screened for the selection of new affinity adsorbents towards biological targets. As a proof of concept, the Petasis-Ugi reaction was here employed in the discovery of affinity ligands suitable for phosphopeptide enrichment. A combinatorial library of 84 ligands was designed, synthesized on a chromatographic solid support and screened in situ for the specific binding of phosphopeptides binding human BRCA1C-terminal domains. The success of the reaction on the chromatographic matrix was confirmed by both inductively coupled plasma atomic emission spectroscopy and fluorescence microscopy. Three lead ligands were identified due to their superior performance in terms of binding capacity and selectivity towards the phosphorylated moiety on peptides, which showed the feasibility of the Petasis-Ugi reaction for affinity ligand development. PMID:27469904

  14. Protein Complex Purification by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, permitting access to target macromolecular complexes thus released in solution. The complexes are specifically enriched from the extract onto a solid medium coupled with an affinity reagent-usually an antibody-that recognizes the target either directly or through an appended affinity tag, allowing subsequent characterization of the complex. Here, we discuss approaches and considerations for purifying endogenous yeast protein complexes by affinity capture. PMID:27371601

  15. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  16. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  17. Compact noncontraction semigroups of affine operators

    NASA Astrophysics Data System (ADS)

    Voynov, A. S.; Protasov, V. Yu

    2015-07-01

    We analyze compact multiplicative semigroups of affine operators acting in a finite-dimensional space. The main result states that every such semigroup is either contracting, that is, contains elements of arbitrarily small operator norm, or all its operators share a common invariant affine subspace on which this semigroup is contracting. The proof uses functional difference equations with contraction of the argument. We look at applications to self-affine partitions of convex sets, the investigation of finite affine semigroups and the proof of a criterion of primitivity for nonnegative matrix families. Bibliography: 32 titles.

  18. Kool-Aid Chromatography

    ERIC Educational Resources Information Center

    Jenkins, Christie L.

    1986-01-01

    Offers guidelines and suggests activities that can introduce middle school students to the process and principles of chromatography in an inexpensive and safe manner. Proposes that experiences with Kool-aid and food coloring chromatography can provide insights into how scientists think, work, and communicate. (ML)

  19. Column Liquid Chromatography.

    ERIC Educational Resources Information Center

    Majors, Ronald E.; And Others

    1984-01-01

    Reviews literature covering developments of column liquid chromatography during 1982-83. Areas considered include: books and reviews; general theory; columns; instrumentation; detectors; automation and data handling; multidimensional chromatographic and column switching techniques; liquid-solid chromatography; normal bonded-phase, reversed-phase,…

  20. Advanced proteomic liquid chromatography

    SciTech Connect

    Xie, Fang; Smith, Richard D.; Shen, Yufeng

    2012-10-26

    Liquid chromatography coupled with mass spectrometry is the predominant platform used to analyze proteomics samples consisting of large numbers of proteins and their proteolytic products (e.g., truncated polypeptides) and spanning a wide range of relative concentrations. This review provides an overview of advanced capillary liquid chromatography techniques and methodologies that greatly improve separation resolving power and proteomics analysis coverage, sensitivity, and throughput.