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Free flow electrophoresis separation and AMS quantitation of 14C-naphthalene-protein adducts  

NASA Astrophysics Data System (ADS)

Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose-(concentration)dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 ?Ci) of 14C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 h post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with 14C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.

Buchholz, Bruce A.; Haack, Kurt W.; Sporty, Jennifer L.; Buckpitt, Alan R.; Morin, Dexter



Free flow electrophoresis separation and AMS quantitation of 14C-naphthalene-protein adducts  

PubMed Central

Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose- (concentration) dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 ?Ci) of 14C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2 D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 hr post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5–11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with 14C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.

Buchholz, Bruce A.; Haack, Kurt W.; Sporty, Jennifer L.; Buckpitt, Alan R.; Morin, Dexter



Comparison of Bromcresol Green and Agarose Protein Electrophoresis for Quantitation of Serum Albumin in Multiple Myeloma  

Microsoft Academic Search

Background: The International Staging System for mul- tiple myeloma has increased the importance of accurate measurement of serum albumin. Two common albumin assays, bromcresol green (BCG) and agarose gel protein electrophoresis (PEL), frequently yield discordant re- sults, creating confusion regarding which assay is supe- rior for use in myeloma. Methods: We measured albumin by BCG on a Roche Modular system,

Christine L. H. Snozek; Amy K. Saenger; Philip R. Greipp; Sandra C. Bryant; Robert A. Kyle; S. Vincent Rajkumar; Jerry A. Katzmann



Determination of biotin on a protein by quantitative sodium dodecyl sulfate–capillary gel electrophoresis of monomeric avidin  

Microsoft Academic Search

Sodium dodecyl sulfate–capillary gel electrophoresis (SDS–CGE) is performed to quantify monomeric avidin and biotin on a protein. Under non-reducing SDS–CGE conditions, avidin migrates as monomers exhibiting apparent molecular mass 17?000. In the presence of a biotin–protein conjugate, monomeric avidin binds the conjugate and forms a larger complex that migrates later in the separation. The difference between the remaining monomeric avidin

Huey G. Lee; Edward Fritsche




SciTech Connect

Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.




Non-denaturating isoelectric focusing gel electrophoresis for uranium-protein complexes quantitative analysis with LA-ICP MS.  


A non-denaturating isoelectric focusing (ND-IEF) gel electrophoresis protocol has been developed to study and identify uranium (U)-protein complexes with laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS) and electrospray ionization mass spectrometry (ESI-MS). The ND-IEF-LA-ICP MS methodology set-up was initiated using in vitro U-protein complex standards (i.e., U-bovine serum albumin and U-transferrin) allowing the assessment of U recovery to 64.4?±?0.4 %. This methodology enabled the quantification of U-protein complexes at 9.03?±?0.23, 15.27?±?0.36, and 177.31?±?25.51 nmol U L(-1) in digestive gland cytosols of the crayfish, Procambarus clarkii, exposed respectively to 0, 0.12, and 2.5 ?mol of waterborne depleted U L(-1) during 10 days. ND-IEF-LA-ICP MS limit of detection was 19.3 pmol U L(-1). Elemental ICP MS signals obtained both in ND-IEF electropherograms and in size exclusion chromatograms of in vivo U-protein complexes revealed interactions between U- and Fe- and Cu-proteins. Moreover, three proteins (hemocyanin, pseudohemocyanin-2, and arginine kinase) out of 42 were identified as potential uranium targets in waterborne-exposed crayfish cytosols by microbore reversed phase chromatography coupled to molecular mass spectrometry (µRPC-ESI-MS/MS) after ND-IEF separation. PMID:23665639

Xu, Ming; Frelon, Sandrine; Simon, Olivier; Lobinski, Ryszard; Mounicou, Sandra



Protein electrophoresis - serum  


Total protein: 6.4 to 8.3 g/dL Albumin: 3.5 to 5.0 g/dL Alpha-1 globulin: 0.1 to 0.3 ... Consumptive coagulopathy Disseminated intravascular coagulation Increased gamma ... Acute infection Waldenstrom's macroglobulinemia ...


Microchip-based capillary electrophoresis of human serum proteins  

Microsoft Academic Search

The separation and relative quantitation of human serum proteins is important to the clinical diagnosis of various states of disease. Microchip-based capillary electrophoresis (CE) of human serum proteins offers several advantages over sodium dodecyl sulfate-poly(acrylamide) gel electrophoresis and conventional CE methods, including decreased sample consumption and analysis time and the possibility of on-chip sample manipulation (dilution, labelling, etc.). The microchip

Christa L. Colyer; Shakuntala D. Mangru; D. Jed Harrison



Quantitation of Serum Free Light Chains in Combination with Protein Electrophoresis and Clinical Information for Diagnosing Multiple Myeloma in a General Hospital Population  

Microsoft Academic Search

BACKGROUND: Serum free light chain (SFLC) measure- ments have recently come into use as an aid for diag- nosing monoclonal gammopathy. We evaluated SFLC measurements in combination with serum protein electrophoresis (SPE) and clinical information for di- agnosing multiple myeloma (MM) in a hospital population.

Armin P. Piehler; Nina Gulbrandsen; Peter Kierulf; Petter Urdal


Automated serum protein electrophoresis by Capillarys.  


Capillary zone electrophoresis (CZE) of serum proteins is increasingly gaining impact in clinical laboratories. In this report, we evaluate automated capillary zone electrophoresis by Capillarys (Sebia, France). Within-run and between-run imprecision for the five electrophoretic fractions was <2% and <6%, respectively. Data obtained with Capillarys correlated with results obtained with agarose gel electrophoresis and Paragon CZE 2000 (Beckman Coulter, USA). Analysis of serum obtained from patients with inflammation, nephrotic syndrome, bisalbuminemia, and alpha1-antitrypsin deficiency revealed that Capillarys was able to detect these abnormalities. Two hundred thirty eight samples were analyzed by agarose gel electrophoresis, Capillarys, capillary electrophoresis using Paragon CZE 2000 system, and immunofixation. Sample selection was based on the presence of a disturbed morphology (e.g., spike) of the protein profile or hypogammaglobulinemia on agarose gel electrophoresis and/or Capillarys. Immunofixation revealed the presence of a monoclonal protein, oligoclonal bands, polyclonal pattern, and a normal profile in, respectively, 89, 66, 19, and 64 samples. With Capillarys, Paragon, and agarose gel electrophoresis, a spike and/or disturbed morphology of the profile was found in 222, 182, and 180 samples, respectively. In these samples, immunofixation was negative in 73 (33%), 46 (25%), and 39 (22%) samples, respectively. These data indicate that Capillarys has a lower specificity than agarose gel electrophoresis and Paragon 2000. Of the 89 samples with a monoclonal protein, Capillarys, Paragon, and agarose gel electrophoresis failed to detect, respectively, three, three, and one monoclonal protein(s). Interferences by radio-opaque agents, complement degradation products, fibrinogen, and triglycerides are described. In conclusion, automated capillary zone electrophoresis with Capillarys provides for reproducible, rapid, and reliable serum electrophoresis. PMID:12812271

Bossuyt, Xavier; Lissoir, Bénédicte; Mariën, Godelieve; Maisin, Diane; Vunckx, Jozef; Blanckaert, Norbert; Wallemacq, Pierre



Relative Quantitative Comparisons of the Extracellular Protein Profiles of Staphylococcus aureus UAMS-1 and Its sarA, agr, and sarA agr Regulatory Mutants Using One-Dimensional Polyacrylamide Gel Electrophoresis and Nanocapillary Liquid Chromatography Coupled with Tandem Mass Spectrometry  

Microsoft Academic Search

One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography cou- pled with mass spectrometry was used to analyze proteins isolated from Staphylococcus aureus UAMS-1 after 3, 6, 12, and 24 h of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more

Richard C. Jones; Joanna Deck; Ricky D. Edmondson; Mark E. Hart



A quantitative method for blood lipoproteins using cellulose acetate electrophoresis  

PubMed Central

A rapid, inexpensive, and quantitative method is described for obtaining the levels of plasma very low, low, and high density lipoproteins using cellulose acetate electrophoresis and lipid assays without prior separation by ultracentrifuge or other techniques. It involves separation of the lipoproteins by cellulose acetate electrophoresis, followed by their identification with the ozone-Schiff reaction. The total lipoprotein concentration is estimated from the total plasma phospholipid, and the percentage of each component obtained by densitometric analysis of the stained electrophoretograms, using reflected light. For samples with a raised level of very low density lipoprotein, plasma triglyceride analysis is also required. The results obtained by the cellulose acetate electrophoresis method are in good agreement with those by the analytical ultracentrifuge and the preparative ultracentrifuge with refractometry. The theoretical assumptions on which the method is based have been shown to be valid. Images

Magnani, H. N.; Howard, A. N.



Using Gel Electrophoresis To Illustrate Protein Diversity and Isoelectric Point.  

ERIC Educational Resources Information Center

|Demonstrates the differences in protein structures by focusing on isoelectric point with an experiment that is observable under certain pH levels in gel electrophoresis. Explains the electrophoresis procedure and reports results of the experiments. (YDS)|

Browning, Mark; Vanable, Joseph



Protein gel electrophoresis in the undergraduate physics laboratory  

Microsoft Academic Search

We describe an undergraduate laboratory experiment in protein gel electrophoresis that uses readily available apparatus and materials. The separation of a mixture of stained proteins by gel electrophoresis was videotaped. Position-time data for the proteins generated from analysis of digitized videotape images allowed for calculation of protein terminal velocities. The dependence of protein terminal velocity on molar mass was determined

Danny G. Miles; David W. Bushman; Zhong-Ying Chen



Serum protein electrophoresis in 147 dogs.  


Reference intervals for serum protein electrophoresis (SPE) were created from a group of 75 clinically healthy dogs and compared with SPE results obtained from clinical cases presented to the University of Bristol over an eight-and-a-half-year period. A total of 147 dogs, in which SPE had been performed, had complete case records available and thus met the inclusion criteria. Signalment and final diagnoses taken from the case records and SPE results were divided into normal and abnormal based on the newly established reference intervals. Cases were grouped according to the SPE protein fraction abnormalities and diagnosis using the DAMNITV classification system. Of the 147 cases, 140 (95.2 per cent) had abnormal SPE results. The most common protein fraction abnormality was decreased albumin (59.3 per cent) followed by a polyclonal increase in ? globulins (38.6 per cent). Decreased ?-1 globulins and increased ?-2 globulins were documented in 36.4 and 30.0 per cent of cases, respectively. The most common DAMNITV classification associated with abnormal SPE results was infectious/inflammatory disease, which was diagnosed in 79 of 140 cases (56.4 per cent). Monoclonal gammopathies were noted in eight dogs (5.7 per cent), and underlying lymphoproliferative disease was present in all cases where a diagnosis was achieved, including multiple myeloma (four dogs), splenic plasmacytoma (one dog), hepatic plasmacytoma (one dog) and lymphoma (one dog). PMID:21493443

Tappin, S W; Taylor, S S; Tasker, S; Dodkin, S J; Papasouliotis, K; Murphy, K F



Processing of DNA and Protein Electrophoresis Gels by Image Analysis  

Microsoft Academic Search

With recent legislation allowing for the registration of new cultivars, the analysis of DNA and protein electrophoresis gels is becoming increasingly important for cultivar identification. DNA fragments or proteins of different molecular weights are separated using electrophoresis, giving a series of bands with positions corresponding to the molecular weight. Image analysis of the gels removes much of the subjectivity of

Donald G. Bailey; C. Bruce Christie


Phylogenetic reconstruction of South American felids defined by protein electrophoresis  

Microsoft Academic Search

Phylogenetic associations among six closely related South American felid species were defined by changes in protein-encoding gene loci. We analyzed proteins isolated from skin fibroblasts using two-dimensional electrophoresis and allozymes extracted from blood cells. Genotypes were determined for multiple individuals of ocelot, margay, tigrina, Geoffroy's cat, kodkod, and pampas cat at 548 loci resolved by two-dimensional electrophoresis and 44 allozyme

J. Pecon Slattery; W. E. Johnson; D. Goldman; S. J. O'Brien



Protein gel electrophoresis in the undergraduate physics laboratory  

NASA Astrophysics Data System (ADS)

We describe an undergraduate laboratory experiment in protein gel electrophoresis that uses readily available apparatus and materials. The separation of a mixture of stained proteins by gel electrophoresis was videotaped. Position-time data for the proteins generated from analysis of digitized videotape images allowed for calculation of protein terminal velocities. The dependence of protein terminal velocity on molar mass was determined and found to agree with predictions made by current theory. We also introduce a model that draws on simple physical concepts to help students place the experimental results in context.

Miles, Danny G.; Bushman, David W.; Chen, Zhong-Ying



Blue Native electrophoresis to study mitochondrial and other protein complexes  

Microsoft Academic Search

The biogenesis and maintenance of mitochondria relies on a sizable number of proteins. Many of these proteins are organized into complexes, which are located in the mitochondrial inner membrane. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a method for the isolation of intact protein complexes. Although it was initially used to study mitochondrial respiratory chain enzymes, it can also be

Leo G. J Nijtmans; Nadine S Henderson; Ian J Holt



Predicting interspecific compatibilities in beans (Phaseolus) by seed protein electrophoresis  

Microsoft Academic Search

Seed proteins of 17 wild species of Phaseolus were separated by electrophoresis on SDS polyacrylamide gels. There was very little variation of the protein pattern within most species, while considerable variation among species was evident. Relative interspecific similarities of protein patterns were estimated using Jaccard's similarity index, and a cluster analysis was performed on these values. The resultant dendrogram generally

J. G. Sullivan; G. Freytag



Quantitative polymerase chain reaction using capillary electrophoresis with laser-induced fluorescence detection: Analysis of duck hepatitis B  

Microsoft Academic Search

We report an accurate and reproducible DNA quantitation method using the polymerase chain reaction (PCR). The amount of PCR product is monitored after each PCR cycle by capillary electrophoresis. To ensure accurate quantitation, a non-amplified internal standard is added to each PCR-amplified electrophoresis sample to correct for variations in injection volume. Quantitation of the sample is based on the number

Nan Li; Woei G. Tan; Roger Y. Tsang; David L. J. Tyrrell; Norman J. Dovichi



Triple-spot proteins in two-dimensional gel electrophoresis.  

PubMed Central

A triple-spot pattern of polypeptides occurring in two-dimensional gel electrophoresis of proteins is described. The presence of a mutant protein, Pc 1 Duarte, which results in a splitting of all three polypeptides, is evidence that they are produced by the same gene. This pattern is seen in about 1% of the proteins from a variety of sources. Typically, about 50% of the protein occurs as a single major spot, the remainder occurring as two polypeptides with an additional negative charge and slightly different molecular weight. The reproducibility of this pattern implies a functional significance which is presently unknown. The implication of this configuration for patterns seen by one-dimensional gel electrophoresis is discussed. Images Fig. 1

Comings, D E; Peters, K E



Application of capillary electrophoresis for DNA-protein binding tests  

Microsoft Academic Search

The combination of affinity chromatography and capillary electrophoresis (CE-SDS) has been found to be a useful tool to analyse\\u000a populations of proteins which specifically bind to ssor dsDNA. Proteins were extracted from tissue, cytosol or nuclei of meristems\\u000a of Pisum sativum seedlings and separated on cellulose column functionalized with ss-, dsDNA (calf thymus) and ssDNA (P. sativum) at 2M concentration

Andrzej Ka?mierczak; Joanna M. Ka?mierczak



Analysis of protein glycation using phenylboronate acrylamide gel electrophoresis  

Microsoft Academic Search

The incorporation of the specialized carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for SDS-PAGE analysis has been successful for the separation of carbohydrates and has here been adapted for the analysis of post-translationally modified proteins. While conventional SDS-PAGE analysis cannot distinguish between glycated and unglycated proteins, methacrylamido phenylboronate acrylamide gel electrophoresis (mP-AGE) in low loading shows dramatic retention

Marta P. Pereira Morais; Julia D. Mackay; Savroop K. Bhamra; J. Grant Buchanan; T. D. James; John S. Fossey; Jean M. H. van den Elsen



Two-dimensional electrophoresis and computer imaging: quantitation of human milk casein.  


Because human casein does not precipitate from milk at its isoelectric point as does bovine casein, there is no easy method of quantitation. Casein represents only approximately 30% of the protein fraction in human milk, and the complex methods necessary for isolation cannot be used easily with small samples in a survey of a large number of mothers. Two-dimensional electrophoresis coupled with computer imaging has the potential to compare and quantitate proteins expeditiously using a small sample size. IsoDalt, a denaturing methodology, separates the casein micelle into its component parts, beta-casein, kappa-casein, parakappa-casein and casomorphins. Identification of these spots was made by immunoassay of a Western blot with monoclonal anti-human casein. Two spots at 24 kDa and 26 kDa, thought to be phosphorylated isomers of beta casein, were selected for quantitation. Milk samples from 20 mothers, 8 weeks post partum, were run on two-dimensional (2-D) gels; a slide was taken of each silverstained gel with a Kodak control strip; the slide was scanned into powerMac Photoshop 3 with a Polaroid-Sprintscan; spots were isolated using "threshold", "mask" with IPTK (Imaging Processing Tool Kit, Reindeer Games) a Photoshop plug-in, and transferred to the NIH-Image program. Using an NIH-Image gel macro (Thomas Seebacher), the area and integrated density of the spots were measured. The Kodak control scale provided calibration and conversion to OD units. Visual scanning of the gels and computer units indicated a wide range of concentrations. To understand the range in units of weight, a standard was generated using bovine alpha casein (Sigma). Measurements will be used in a statistical program, Statview (Abacus), in an attempt to correlate information from a questionaire with casein concentration. PMID:10344261

Goldfarb, M


Chapter 8 Quantitation of Protein  

Microsoft Academic Search

The measurement of protein concentration in an aqueous sample is an important assay in biochemistry research and development labs for applications ranging from enzymatic studies to providing data for biopharmaceutical lot release. Spectrophotometric protein quantitation assays are methods that use UV and visible spectroscopy to rapidly determine the concentration of protein, relative to a standard, or using an assigned extinction

James E. Noble; Marc J. A. Bailey



Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin.  


The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms. PMID:24120174

Marie, Anne-Lise; Przybylski, Cédric; Gonnet, Florence; Daniel, Régis; Urbain, Rémi; Chevreux, Guillaume; Jorieux, Sylvie; Taverna, Myriam



Human muscle proteins: analysis by two-dimensional electrophoresis  

SciTech Connect

Proteins from single frozen sections of human muscle were separated by two-dimensional gel electrophoresis and detected by fluorography or Coomassie Blue staining. The major proteins were identical in different normal muscles obtained from either sex at different ages, and in Duchenne and myotonic dystrophy samples. Congenital myopathy denervation atrophy, polymyositis, and Becker's muscular dystrophy samples, however, showed abnormal myosin light chain compositions, some with a decrease of fast-fiber myosin light chains and others with a decrease of slow-fiber light chains. These protein alterations did not correlate with any specific disease, and may be cause by generalized muscle-fiber damage.

Giometti, C.S.; Danon, M.J.; Anderson, N.G.



Quantitation of Plasma Membrane Calcium Pump Phosphorylated Intermediates by Electrophoresis  

Microsoft Academic Search

P-ATPases are characterized by the formation of acid-stable phosphorylated intermediates (EP) during their reaction cycle. We have developed a microscale method to determine EP that involves the phosphorylation of the enzyme using [?-32P]ATP and precipitation with TCA; separation of the sample by SDS-PAGE, and measurement of the enzyme protein and 32P-labeled EP by digital analysis of both the stained gel

Mar??a Mercedes Echarte; Valeria Levi; Ana Mar??a Villamil; Rolando C. Rossi; Juan Pablo F. C. Rossi



High Resolution Two-Dimensional Electrophoresis of Proteins*  

PubMed Central

Summary A technique has been developed for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis. Due to its resolution and sensitivity, this technique is a powerful tool for the analysis and detection of proteins from complex biological sources. Proteins are separated according to isoelectric point by isoelectric focusing in the first dimension, and according to molecular weight by sodium dodecyl sulfate electrophoresis in the second dimension. Since these two parameters are unrelated, it is possible to obtain an almost uniform distribution of protein spots across a two-dimensional gel. This technique has resolved 1100 different components from Escherichia coli and should be capable of resolving a maximum of 5000 proteins. A protein containing as little as one disintegration per min of either 14C or 35S can be detected by autoradiography. A protein which constitutes 10?4 to 10?5% of the total protein can be detected and quantified by autoradiography. The reproducibility of the separation is sufficient to permit each spot on one separation to be matched with a spot on a different separation. This technique provides a method for estimation (at the described sensitivities) of the number of proteins made by any biological system. This system can resolve proteins differing in a single charge and consequently can be used in the analysis of in vivo modifications resulting in a change in charge. Proteins whose charge is changed by missense mutations can be identified. A detailed description of the methods as well as the characteristics of this system are presented.

O'Farrell, Patrick H.



Procedures for two-dimensional electrophoresis of proteins  

SciTech Connect

High-resolution two-dimensional gel electrophoresis (2DE) of proteins, using isoelectric focusing in the first dimension and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) in the second, was first described in 1975. In the 20 years since those publications, numerous modifications of the original method have evolved. The ISO-DALT system of 2DE is a high-throughput approach that has stood the test of time. The problem of casting many isoelectric focusing gels and SDS-PAGE slab gels (up to 20) in a reproducible manner has been solved by the use of the techniques and equipment described in this manual. The ISO-DALT system of two-dimensional gel electrophoresis originated in the late 1970s and has been modified many times to improve its high-resolution, high-throughput capabilities. This report provides the detailed procedures used with the current ISO-DALT system to prepare, run, stain, and photograph two-dimensional gels for protein analysis.

Tollaksen, S.L.; Giometti, C.S.



Two-dimensional acrylamide gel electrophoresis of cancer-patient serum proteins.  


Sera from normal volunteers, patients with a variety of non-neoplastic diseases and patients with malignant or benign tumors were examined by two-dimensional acrylamide gel electrophoresis. In this technique the serum is first separated in an acrylamide gel column followed by a second electrophoresis at right angles to the first separation in a continuous concave 2 to 30 percent gradient acrylamide gel slab. The stained two-dimensional gel slab appears as a "fingerprint" pattern or "map" of the separated serum proteins. Both qualitative and quantitative differences in the fingerprint patterns of cancer-patient sera were observed. The quantitative alterations did not appear specifically associated with malignant tumors. However, several qualitative differences were detected, of which some may represent markers of malignancy as they were not observed in the normal, abnormal and benign tumors control sera. At least one of the abnormal protein stained spots, a prealbumin, appears to be restricted and related in some way to cancer of the lymphoreticular system. These data, although limited, support earlier indications that two-dimensional acrylamide gel electrophoresis offers a new and powerful tool to the cancer scientist for the detection of alterations in cancer-patient sera. A discussion of its possible use to examine other biological fluid and tumor extracts from the cancer patient is presented. PMID:4376659

Wright, G L


High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture(SILAC), two-dimensional gel electrophoresis(2DE) and nano-liquid chromatograpohy coupled to an LTQ-OrbitrapMass spectrometer.  


The proteomics field has shifted over recent years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel-free workflows because of the tremendous developments in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry. However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined stable isotope labeling with amino acids in cell culture of controls and apoptotic HeLa cells with 2-DE and the subsequent analysis of tryptic peptides via nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer to obtain quantitative data using the methods with the highest resolving power on all levels of the proteomics workflow. More than 1,200 proteins with more than 2,700 protein species were identified and quantified from 816 Coomassie Brilliant Blue G-250 stained 2-DE spots. About half of the proteins were identified and quantified only in single 2-DE spots. The majority of spots revealed one to five proteins; however, in one 2-DE spot, up to 23 proteins were identified. Only half of the 2-DE spots represented a dominant protein with more than 90% of the whole protein amount. Consequently, quantification based on staining intensities in 2-DE gels would in approximately half of the spots be imprecise, and minor components could not be quantified. These problems are circumvented by quantification using stable isotope labeling with amino acids in cell culture. Despite challenges, as shown in detail for lamin A/C and vimentin, the quantitative changes of protein species can be detected. The combination of 2-DE with high-resolution nano-liquid chromatography-mass spectrometry allowed us to identify proteomic changes in apoptotic cells that would be unobservable using any of the other previously employed proteomic workflows. PMID:23033477

Thiede, Bernd; Koehler, Christian J; Strozynski, Margarita; Treumann, Achim; Stein, Robert; Zimny-Arndt, Ursula; Schmid, Monika; Jungblut, Peter R



Numerical simulation of protein separation by continuous-flow electrophoresis.  


Continuous-flow electrophoresis is a process for separating protein mixtures on a preparative scale. Its resolution is determined by the migration distance at the collection plane and by the fineness of the filament occupied by each protein species. Filaments undergo spreading due to a number of different phenomena, among which electrokinetics and electrohydrodynamics are known to be important. In the first of these, differences in migration velocity between the ionic species give rise to local variations in pH and electrical conductivity near the protein filament. In the second, the local change in electrical conductivity distorts the electric field, thus inducing shear stress in the liquid and creating a local flow pattern. A numerical model has been developed to describe these phenomena when two proteins are being separated. PMID:8137791

Clifton, M J



Quantitative analysis of low molecular weight carboxylic acids by capillary zone electrophoresis\\/conductivity detection  

Microsoft Academic Search

Low molecular weight carboxylic acids are separated and quantitated by capillary zone electrophoresis (CZE) with an on-column conductivity detector. The addition of 0.2-0.5 mM TTAB (tetradecyltrimethylammonium bromide) controls the electroosmotic flow so that all carboxylate anions pass through the detector. Unlike other CZE detection methods, conductivity detection shows a direct relationship between retention time and peak area. This confers on

X. Huang; J. A. Luckey; M. J. Gordon; R. N. Zare



Meat species identification by linear discriminant analysis of capillary electrophoresis protein profiles.  


The objective of this study was to utilize linear discriminant analysis (LDA) in the interpretation of capillary electrophoresis-sodium dodecyl sulfate polymer-filled capillary gel electrophoresis (CE-SDS) meat protein profiles for the identification of meat species. The specific objectives were 1) to collect quantitative data on water-soluble and saline-soluble proteins of different meat species obtained by CE-SDS and 2) to apply LDA on collected CE-SDS protein data for the development of a pattern recognition statistical model useful in the differentiation of meat species. Samples were raw beef top and eye round, boneless fresh pork ham and loin, turkey leg and breast meat, and mechanically deboned turkey meat collected on six different occasions, making a total of 42 samples. Additionally, 14 samples were used as test samples to determine the classification ability of the procedure. Quantitative protein data obtained by CE-SDS was used to generate separate LDA models for either water- or saline-soluble protein extracts. Although a saline solution was a more efficient meat protein-extracting agent, as shown by a higher total protein concentration and a larger number of peaks, water-soluble CE-SDS protein profiles gave more distinctive discrimination among meat species. The correct classification given by LDA on water-soluble protein data was 100% for all meat species, except pork (94%). Conversely, the correct classification on saline-soluble protein data was 88% for beef and mechanically deboned turkey meat, and 94% and 100% for turkey and pork meat, respectively. LDA proved to be a useful pattern recognition procedure in the interpretation of CE-SDS protein profiles for the identification of meat species. PMID:10812424

Vallejo-Córdoba, B; Cota-Rivas, M


Quantitative aspects of rare earth metal determinations using capillary electrophoresis with indirect absorbance detection  

SciTech Connect

The practical utility of capillary zone electrophoresis with indirect absorbance detection is examined for the separation and quantitation of rare earth metals. Various imidazole derivatives are investigated as to their suitability as running buffer (displaceable) detection ions with {alpha}-hydroxyisobutyric acid functioning as a chelating agent to enhance separations. Parameters important for quantitative analysis, such as limits of detection, relative standard deviation of peak areas, efficiency, resolution, peak shape and linear dynamic range are presented. The influence of sample matrix, method of injection, and background ion identity on these parameters are investigated and discussed.

Colburn, B.A.; Starnes, S.D.; Sepaniak, M.J. [Univ. of Tennessee, Knoxville, TN (United States)] [and others



Identification of M protein from filter paper using serum protein and immunofixation electrophoresis.  


Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are standard methods for detection and monitoring of monoclonal (M) proteins. However, these tests are rarely available in the remote areas, especially in developing countries. Transportation of fresh serum (FS) samples is also usually inconvenient. This study investigated M-protein identification using serum blot on filter paper (FP). SPE and IFE were performed on FS and FP specimens using the Sebia Hydrasys automated electrophoresis system. Statistical analyses were conducted to assess sample stability and agreement of FS vs FP. The FP method showed good agreement with the FS method. The r values for correlation of albumin levels-?(1), ?(2), ?, and ? (%)-between FP and FS samples in SPE were all more than 0.95 (P < .01). IFE displayed no significant difference between those 2 methods in the identification of M protein. The FP method demonstrated an accurate and reproducible alternative to FS for identification of M protein using SPE and IFE. PMID:23010716

Wu, Yonghua; Yang, Xu; Wang, Tiancheng; Wang, Haining; Li, Zhenrong



Quantitative determination of alginic acid in pharmaceutical formulations using capillary electrophoresis.  


A capillary electrophoresis (CE) method has been developed and validated for the quantitative determination of alginic acid, which is used as a rafting agent in complex antacid formulations. The method involves a preliminary separation of the alginic acid from the formulation by washing the sample matrix with methanol, diluted HCl and water. This is followed by electrophoresis within a fused silica capillary using borate/boric acid buffer as the electrolyte, and the quantification is performed by a UV detector monitoring at 200 nm, where the intrinsic absorption of alginic acid is measured. An assay precision of better than 3% was achieved in intra- and interday determinations. No interference was found from the matrix of the antacid formulations. PMID:14738939

Moore, Douglas E; Miao, William G; Benikos, Con



Serum Protein Electrophoresis in the Evaluation of Lytic Bone Lesions  

PubMed Central

Serum protein electrophoresis (SPEP) is often obtained at the initial evaluation of a radiolucent bone lesion of unknown etiology. The results are considered convincing evidence of the presence or absence of a plasma cell neoplasm. The sensitivity and specificity of the SPEP have not been reported in this clinical scenario. Our purpose is to assess the diagnostic value of the SPEP in the initial work-up of the radiolucent bone lesion. We identified 182 patients undergoing evaluation of a radiolucent bone lesion that included tissue biopsy and an SPEP value. We then calculated the sen-sitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of SPEP as a diagnostic test for a plasma cell neo-plasm in this clinical scenario. Forty-six of 182 (25.3%) patients in our series were diagnosed with a plasma cell neo-plasm by histopathologic analysis. The sensitivity of SPEP was 71% and the specificity was 83%. PPV was 47% and NPV was 94%. When analyzing only those presenting with multiple lesions, the percentage of patients diag-nosed with multiple myeloma increased to 44.7% (34 of 76 patients). The SPEP, however, did not have a substantially increased diagnostic accuracy with sensitivity of 71%, specificity 79%, PPV 40% and NPV 93%. SPEP lacks sensitivity and positive predictive value to provide a definitive diagnosis of myeloma in radiolucent bone lesions, but has a high negative predictive value which may make it useful in ruling out the disease. We recommend that this test either be performed in conjunction with urine electrophoresis, immunofixation electro-phoresis and free light chain assay, or after biopsy confirming the diagnosis of myeloma.

Nystrom, Lukas M.; Buckwalter, Joseph A.; Syrbu, Sergei; Miller, Benjamin J.



Serum protein electrophoresis in the evaluation of lytic bone lesions.  


Serum protein electrophoresis (SPEP) is often obtained at the initial evaluation of a radiolucent bone lesion of unknown etiology. The results are considered convincing evidence of the presence or absence of a plasma cell neoplasm. The sensitivity and specificity of the SPEP have not been reported in this clinical scenario. Our purpose is to assess the diagnostic value of the SPEP in the initial work-up of the radiolucent bone lesion. We identified 182 patients undergoing evaluation of a radiolucent bone lesion that included tissue biopsy and an SPEP value. We then calculated the sen-sitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of SPEP as a diagnostic test for a plasma cell neo-plasm in this clinical scenario. Forty-six of 182 (25.3%) patients in our series were diagnosed with a plasma cell neo-plasm by histopathologic analysis. The sensitivity of SPEP was 71% and the specificity was 83%. PPV was 47% and NPV was 94%. When analyzing only those presenting with multiple lesions, the percentage of patients diag-nosed with multiple myeloma increased to 44.7% (34 of 76 patients). The SPEP, however, did not have a substantially increased diagnostic accuracy with sensitivity of 71%, specificity 79%, PPV 40% and NPV 93%. SPEP lacks sensitivity and positive predictive value to provide a definitive diagnosis of myeloma in radiolucent bone lesions, but has a high negative predictive value which may make it useful in ruling out the disease. We recommend that this test either be performed in conjunction with urine electrophoresis, immunofixation electro-phoresis and free light chain assay, or after biopsy confirming the diagnosis of myeloma. PMID:24027470

Nystrom, Lukas M; Buckwalter, Joseph A; Syrbu, Sergei; Miller, Benjamin J



Protein Electrophoresis in the Biology Classroom Using "Safe" Gels.  

ERIC Educational Resources Information Center

|Describes the use of electrophoresis in the high school utilizing two new gels that are easy to pour, do not require degassing, can be used on a horizontal gel electrophoresis, and are not neurotoxic or carcinogenic health hazards. Large diagrams and written instructions are used to present the protocol of setting up the electrophoresis. (PR)|

Atkins, Thomas



Factors affecting quantitative electrokinetic injections from submicroliter conductive vials in capillary electrophoresis.  


The factors influencing quantitative electrokinetic injections in capillary electrophoresis for custom 340-nL, 10-microL, and 110-microL stainless steel sample vials have been investigated using a six-analyte mixture containing catecholamines and indolamines. Deleterious sample degradation is increased with smaller sampling vials, decreased capillary-electrode distances, and increased current passed during the injection. Zero-voltage injections from the smallest vials also demonstrate additional injection discrepancies when compared to larger-volume bulk solution injections. These effects are in addition to the electrokinetic bias and complicate the selection of appropriate internal standards. For nanoliter-volume conductive vials, the injection process creates new species and eliminates other electroactive species to such an extent that quantitation becomes problematic. PMID:10500488

Fuller, R R; Sweedler, J V



Quantitative determination of vasicine and vasicinone in Adhatoda vasica by high performance capillary electrophoresis.  


A new method of capillary electrophoresis was developed for the quantitative determination of vasicine and vasicinone from Adhatoda vasica (L.) Nees. The electrophoretic separation was performed using a 47 cm x 50 microm ID (38.5 cm effective length) fused silica capillary. The samples were injected by pressure for 3 s at 50 mbar and the running voltage was 19 kV at the injector end of the capillary. The capillary temperature was maintained at 40 degrees C. The separation of the two alkaloids has been achieved within 11 min with good repeatability. The method was validated in terms of reproducibility, linearity, accuracy and applied for the quantitative determination of vasicine and vasicinone in A. vasica plant samples/extracts. Parameters affecting the resolution such as pH, temperature, organic modifier, buffer concentration and capillary dimensions were reported. PMID:18271297

Avula, B; Begum, S; Ahmed, S; Choudhary, M I; Khan, I A



Attomole quantitation of protein separations with accelerator mass spectrometry  

SciTech Connect

Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to sub-attomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5% . Micro-proton-induced-xray-emission quantifies elemental abundances in separated metalloprotein samples to nanogram amounts and is capable of quantifying phosphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.

Vogel, J S; Grant, P G; Buccholz, B A; Dingley, K; Turteltaub, K W



Plaque quantitation through protein measurement.  


This study was undertaken to establish whether the quantitation of dental plaque protein by a dye-binding method (Coomassie G-250) may be used as an index of the amount of dental plaque sampled. Ten sites were sampled in 34 children on 5 occasions at 4 month intervals. The mean protein concentration in 1391 plaque samples was 6.9 +/- 4.1 micrograms (micrograms) (mean +/- standard deviation). A three-way analysis of variance showed that the plaque protein concentration was similar at the different sampling sites in the same child (p = 0.14), but statistically significant differences were observed with respect to time of sampling (F = 36.24; p = 0.0001) and individual sampled (F = 5.69; p = 0.0001). These observations indicate that plaque bacterial counts may be expressed as units of protein concentration and this method may be useful to relate the number of viable bacteria to an estimate of the amount of plaque collected. This ratio allows standardisation for any variation in the amount of plaque collected. PMID:1401432

Smit, A; Cleaton-Jones, P E; Boardman, M E



Identification of fibrosis-relevant proteins using DIGE (difference in gel electrophoresis) in different models of hepatic fibrosis.  


Proteomics became a more and more important technique for the large-scale analysis of proteins during the last years. Two-dimensional (2D) electrophoresis as a major tool of proteomics is a powerful method to compare two different biological stages (e. g. healthy and diseased tissue) and to find differences in their protein pattern. One major problem in proteomics is the gel to gel variation of two-dimensional gel electrophoresis, which could cause artefacts in the detection of expression differences. The "difference in gel electrophoresis" (DIGE) technique allows the separation of two proteomes in the same gel. The protein pools were labelled with different fluorescent dyes and equal amounts of protein were separated in the same gel. Another advantage of DIGE is the possibility to separate an internal standard labelled with a third dye in the same gel to allow quantitative expression analysis. We compared proteomes of three different fibrosis models with the appropriate control (tissue inhibitor of metalloproteinases-1 (TIMP-1) overexpressing HepG2 cells in comparison to a HepG2 control, freshly isolated HSC in comparison to activated HSC and healthy mouse liver in comparison to fibrotic mouse liver). Among the differentially expressed proteins several were already found to be relevant for fibrosis but we also detected some proteins like the selenium binding protein 2 which might be relevant for hepatic fibrosis. PMID:15650968

Henkel, C; Roderfeld, M; Weiskirchen, R; Scheibe, B; Matern, S; Roeb, E



High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes.  


Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses. PMID:17426019

Wittig, Ilka; Karas, Michael; Schägger, Hermann



A rapid quantitative determination of phenolic acids in Brassica oleracea by capillary zone electrophoresis.  


A simple and rapid capillary zone electrophoresis method to quantitatively determine the phenolic acid contents in brassica vegetables is described. Phenolic compounds were extracted from broccoli, broccolini, Brussels sprouts, cabbage and cauliflower and the main hydroxycinnamic acids (sinapic, ferulic, p-coumaric and caffeic acids) were isolated by solid phase extraction with C18 cartridges. Using an optimised method, the four analytes were separated in less than 7min in a 50?m×60cm capillary with a 15mM borate buffer (pH=9.13) and a separation voltage of 30kV at 30°C. A linear relationship was observed for the method (r=0.9997-0.9999) with detection limits ranging from 1.1 to 2.3mg/kg of vegetables for the analytes. This method demonstrated good reproducibility with coefficients of variation of less than 5% for peak area and less than 1% for migration time (n=7). The method was successfully applied to quantitatively determine the phenolic acid contents in a range of brassica vegetables and the results were in good agreement when compared to those from high performance liquid chromatography analysis. PMID:23140738

Lee, Iris S L; Boyce, Mary C; Breadmore, Michael C



Analyses of mouse and Drosophila proteins by two-dimensional gel electrophoresis  

Microsoft Academic Search

Two-dimensional gel electrophoresis was employed for the protein analysis of several different mouse tissues and Drosophila. The number of protein spots detected with conventional protein dye staining techniques ranged from 110 in erythrocyte lysate to 320 in liver homogenate. Strain variation of protein spots on the gels was examined in five different tissues from two strains of inbred mice (DBA\\/2J

Chi-Yu Lee; Daniel Charles; Duane Bronson; Michael Griffin; Lawrence Bennett



Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins.  


We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome. PMID:22531133

Goyder, Miriam S; Willison, Keith R; Klug, David R; Demello, Andrew J; Ces, Oscar



Cellulose Acetate Electrophoresis of Cerebrospinal Fluid Proteins with Normal Values of 135 Persons.  

National Technical Information Service (NTIS)

A study on cellulose acetate electrophoresis of cerebrospinal fluid (CSF) proteins is reported. Two methods are used for concentration, acetone precipitation and sephadex dialysis with slight modifications. They are both considered to be clinically valuab...



Cationic electrophoresis.  


Denaturing, discontinuous electrophoresis in the presence of SDS has become a standard method for the protein scientist. However, there are situations where this method produces suboptimal results. In these cases, electrophoresis in the presence of positively charged detergents such as cetyltrimethylammonium bromide (CTAB) may work considerably better. Methods for electrophoresis and staining of such gels are presented. PMID:22585477

Buxbaum, Engelbert



Two-dimensional electrophoresis of liver proteins: characterization of a drug-induced hepatomegaly in rats.  


Two-dimensional electrophoresis (2-DE) of liver proteins was applied to further characterize an unusual drug-induced increase in hepatocellular rough endoplasmic reticulum (RER) in Sprague-Dawley rats given a substituted pyrimidine derivative. Absolute liver weights of drug-treated rats (9.9 +/- 0.4 g) increased above vehicle-treated controls (7.2 +/- 0.2 g) by 37%. Light microscopy revealed diffuse granular basophilia of the hepatocellular cytoplasm, uncharacteristic of hepatocytes and suggested cells rich in ribosomes, which was confirmed by electron microscopy. Immunostaining for cell proliferation, viz., 5-bromo-2'-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA), indicated marked hepatocellular proliferative activity. 2-DE of solubilized liver using an ISO-DALT gel system indicated significant (p<0.001) quantitative changes in at least 17 liver proteins (12 increased, 5 decreased) compared to controls. The protein with the largest increase was homologous to acute-phase reactant, contrapsin-like protein inhibitor-6. Other markedly upregulated proteins were methionine adenosyltransferase, a catalyst in methionine/ATP metabolism and mitochondrial HMG-CoA synthase, involved in cholesterol synthesis. The complementary strategies of 2-DE coupled either with database spot mapping or protein isolation and amino acid sequencing successfully identified a subset of proteins from xenobiotic-damaged rodent livers, the expression of which differed from controls. However, the current bioinformatics platform for rodent hepatic proteins and limited knowledge of specific protein functionality restricted application of this proteomics profile to further define a mechanistic basis for this unusual hepatotoxicity. PMID:10892723

Newsholme, S J; Maleeff, B F; Steiner, S; Anderson, N L; Schwartz, L W




SciTech Connect

This is the final report of a three-year, Laboratory-Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The goal of this project was to try to detect unstained, untagged, unlabeled DNA bands in real-time during gel electrophoresis using simple thermal measurements. The technical and ES&H advantages to this approach could potentially be quite significant, especially given the extreme importance of gel electrophoresis to a wide variety of practical and research fields. The project was unable to demonstrate sufficient thermal sensitivity to detect DNA bands. It is clear that we still do not understand the gel electrophoresis phenomenon very well. The temperature control techniques developed during the course of this project have other useful applications.




Single-cell protein analysis of a single mouse embryo by two-dimensional capillary electrophoresis  

Microsoft Academic Search

The characterization of protein expression from a single-cell mouse embryo using two-dimensional capillary electrophoresis (2D-CE) is described. These zygotes were obtained from Hsf1 gene knockout mice. Single zygotes were lysed off-column and proteins were fluorescently labeled using the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). After injection, analytes were separated first according to molecular weight using capillary sieving electrophoresis (CSE) and then by

Melissa M. Harwood; Elisabeth S. Christians; Norman J. Dovichi



Serum protein fractionation using supported molecular matrix electrophoresis.  


Supported molecular matrix electrophoresis (SMME), in which a hydrophilic polymer such as PVA serves as a support within a porous PVDF membrane, was recently developed. This method is similar to cellulose acetate membrane electrophoresis but differs in the compatibility to glycan analysis of the separated bands. In this report, we describe the first instance of the application of SMME to human serum fractionation, and demonstrate the differences with serum fractionation by cellulose acetate membrane electrophoresis. The SMME membrane exhibited almost no EOF during electrophoresis, unlike the cellulose acetate membrane, but afforded comparative results for serum fractionation. The visualization of each fraction was achieved by conventional staining with dye such as Direct Blue-71, and objective quantification was obtained by densitometry after inducing membrane transparency with 1-nonene. Immunostaining was also achieved. Moreover, mass spectrometric analysis of both N-linked and O-linked glycans from the separated bands was demonstrated. Serum fractionation and glycan profiling of each fraction using SMME will enable novel insights into the relationships between various glycosylation profiles and disease states. PMID:23765906

Dong, Weijie; Matsuno, Yu-Ki; Kameyama, Akihiko



High Resolution Clear Native Electrophoresis for In-gel Functional Assays and Fluorescence Studies of Membrane Protein Complexes  

Microsoft Academic Search

Clear native electrophoresis and blue native electro- phoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic ac- tivity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein

Ilka Wittig; Michael Karas; Hermann Schagger



A quantitative method for measuring protein phosphorylation  

Microsoft Academic Search

We have developed a novel method for quantitating protein phosphorylation by a variety of protein kinases. It can be used with purified kinases and their substrates in vitro or in combination with cell extracts. The method is based on the knowledge that protein kinase C (PKC) adds three phosphates to each molecule of its preferred substrate, myelin basic protein (MBP).

J. Andres Mckenzie; Phyllis R. Strauss



Characterization of ribosomal proteins from different tissues and species of animals by electrophoresis on polyacrylamide gel  

Microsoft Academic Search

Summary Proteins from ribosomes of different tissues and animals were characterized by polyacrylamide disc electrophoresis. The proteins from ribosomes of different tissues from the same animal are qualitatively similar. The results of the experiments with ribosomes from the livers of different species of animals exhibit clear differences, in the electrophoretic patterns of the proteins.

H. Bielka; H. Welfle



Charge Shift Electrophoresis: Simple Method for Distinguishing between Amphiphilic and Hydrophilic Proteins in Detergent Solution  

Microsoft Academic Search

Seventeen hydrophilic proteins and five amphiphilic membrane proteins were subjected to agarose gel electrophoresis in the presence of a nonionic detergent (Triton X-100), a mixture of a nonionic and an anionic detergent (Triton X-100 and sodium deoxycholate), and a mixture of a nonionic and a cationic detergent (Triton X-100 and cetyltrimethylammonium bromide). The electrophoretic mobility of the hydrophilic proteins was

Ari Helenius; Kai Simons



Assay of plant proteins with bicinchoninic acid for high resolution two-dimensional polyacrylamide gel electrophoresis  

Microsoft Academic Search

A method is described for estimating proteins in the same plant tissue sample that is solubilized for separation by two-dimensional polyacrylamide gel electrophoresis. The method uses a modified bicinchoninic acid (BCA) protein assay procedure and a modified standard urea solubilization buffer to estimate microgram values of unknown protein concentration, in the presence of 9 M urea and 4% Nonidet P-40,

Alan R. Orr; Brett A. Wagner; Catherine T. Howard; Orlando A. Schwartz




Technology Transfer Automated Retrieval System (TEKTRAN)

A general procedure for two-dimensional (2-D) gel electrophoresis of Citrus leaves has been developed. We evaluated the resolution of 2-D protein patterns for Tris-HCl and KCl extractions of water soluble proteins and a phenol extraction for extraction of hydrophobic cell membrane proteins from Cha...


Development of a novel strategy for preconcentration of antibiotic residues in milk and their quantitation by capillary electrophoresis.  


A novel analytical method based on capillary zone electrophoresis coupled with diode array detection is developed and validated for the identification and simultaneous quantitation of four antibiotics in bovine raw milk. The studied antibiotics belong to different groups: beta-lactams, tetracyclines, quinolones, amphenicols and sulfonamides. An experimental design including both a factorial and a central composite design allowed a reduction in the number of optimization experiments. The multiple response criterion was successfully used to optimize the separation between chloramphenicol, ciprofloxacin, ampicillin, tetracycline and sulfamethoxazol, allowing the reduction of the analysis time with excellent peak resolutions and low capillary current. Different strategies for preconcentration and extraction of the studied antibiotics were applied, in order to remove potential interferences from the sample and to increase the sensitivity. Milk samples were prepared by a clean-up/extraction procedure based on protein precipitation with trichloroacetic acid followed by liquid-liquid extraction with dichloromethane combined with solid-phase extraction, and injection into the electrophoretic system hydrodynamically. The limits of detection and quantification (below 30 and 100 microg L(-1), respectively) were in all cases lower than the maximum residue limits tolerated for these compounds in milk. Accuracy was evaluated by computing recoveries for the target antibiotics which were between 93.08% and 102.89%. PMID:20685459

Vera-Candioti, Luciana; Olivieri, Alejandro C; Goicoechea, Héctor C



Comparison of selected analytical techniques for protein sizing, quantitation and molecular weight determination  

Microsoft Academic Search

Protein analysis techniques are developing fast due to the growing number of proteins obtained by recombinant DNA techniques. In the present paper we compare selected techniques, which are used for protein sizing, quantitation and molecular weight determination: sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), lab-on-a-chip or microfluidics technology (LoaC), size exclusion chromatography (SEC) and mass spectrometry (MS). We compare advantages and limitations

H. Goetz; M. Kuschel; T. Wulff; C. Sauber; C. Miller; S. Fisher; C. Woodward



Quantitative determination of oxidized carbon nanotube probes in yeast by capillary electrophoresis with laser-induced fluorescence detection  

Microsoft Academic Search

Short oxidized multi-walled carbon nanotubes were functionalized with fluorescein isothiocyanate to form carbon nanotube probes (CNTP). The distribution of CNTP in yeast was quantitatively determined by capillary electrophoresis coupled with laser-induced fluorescence detection. The detection sensitivity for CNTP was greatly improved comparing with UV absorbance and Raman detection. The time- and temperature-dependent influx patterns of CNTP into yeast were obtained.

Hua Xiao; Hanfa Zou; Chensong Pan; Xiaogang Jiang; X. Chris Le; Ling Yang



Biologist's perspective on analytical imaging systems as applied to protein gel electrophoresis  

Microsoft Academic Search

High-resolution two-dimensional polyacrylamide gel electrophoresis entails the separation of proteins in the first dimension according to their charge and in the second dimension according to their relative mobility (RF). The technique is capable of simultaneously resolving thousands of polypeptides as a constellation pattern of spots. Ultimately, the level of success in the analysis of such protein patterns depends upon the

Wayne F. Patton



Analysis of wheat flour proteins related to grain hardness using capillary electrophoresis  

Microsoft Academic Search

A simple and fast capillary electrophoresis method was used for the analysis of wheat flour proteins related to grain hardness. The study has shown that the ratio of the peak areas of two proteins, puroindolines a and b, can be used to distinguish hard and soft varieties of breadwheat. In addition, the results indicate that hard breadwheats can be further

Li Day; Philip Greenwell; Stephen Lock; Helen Brown



The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins.  


Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers. PMID:12872236

Pieper, Rembert; Gatlin, Christine L; Makusky, Anthony J; Russo, Paul S; Schatz, Courtney R; Miller, Stanton S; Su, Qin; McGrath, Andrew M; Estock, Marla A; Parmar, Prashanth P; Zhao, Ming; Huang, Shih-Ting; Zhou, Jeff; Wang, Fang; Esquer-Blasco, Ricardo; Anderson, N Leigh; Taylor, John; Steiner, Sandra



Targeted quantitation of proteins by mass spectrometry.  


Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Key advantages of MRM assays are the ability to target specific peptide sequences, including variants and modified forms, and the capacity for multiplexing that allows analysis of dozens to hundreds of peptides. Different quantitative standardization methods provide options that balance precision, sensitivity, and assay cost. Targeted protein quantitation by MRM and related mass spectrometry methods can advance biochemistry by transforming approaches to protein measurement. PMID:23517332

Liebler, Daniel C; Zimmerman, Lisa J



Targeted Quantitation of Proteins by Mass Spectrometry  

PubMed Central

Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Key advantages of MRM assays are the ability to target specific peptide sequences, including variants and modified forms, and the capacity for multiplexing that allows analysis of dozens to hundreds of peptides. Different quantitative standardization methods provide options that balance precision, sensitivity, and assay cost. Targeted protein quantitation by MRM and related mass spectrometry methods can advance biochemistry by transforming approaches to protein measurement.



Quantitative analysis of chemical warfare agent degradation products in reaction masses using capillary electrophoresis.  


Quantitative methods have been developed for the analysis of chemical warfare agent degradation products in reaction masses using capillary electrophoresis (CE). This is the first report of a systematic validation of a CE-based method for the analysis of chemical warfare agent degradation products in agent neutralization matrixes (reaction masses). After neutralization with monoethanolamine/water, the nerve agent GB (isopropyl methylphosphonofluoridate, Sarin) gives isopropyl methylphosphonic acid (IMPA) and O-isopropyl O'-(2-amino)ethyl methylphosphonate (GB-MEA adduct). The nerve agent GD (pinacolyl methylphosphonofluoridate, Soman), [pinacolyl = 2-(3,3-dimethyl)butyl] produces pinacolyl methylphosphonic acid (PMPA) and O-pinacolyl O'-(2-amino)ethyl methylphosphonate (GD-MEA adduct). The samples were prepared by dilution of the reaction masses with deionized water before analysis by CE/indirect UV detection or CE/conductivity detection. Migration time precision was less than 4.0% RSD for IMPA and 5.0 RSD for PMPA on a day-to-day basis. The detection limit for both IMPA and PMPA is 100 micrograms/L; the quantitation limit for both is 500 micrograms/L. For calibration standards, IMPA and PMPA gave a linear response (R2 = 0.9999) over the range 0.5-100 micrograms/mL. The interday precision RSDs were 1.9, 1.0, and 0.7% for IMPA at 7.5, 37.5 and 75.0 micrograms/mL, respectively. Corresponding values for PMPA (again, RSD) were 2.9, 1.1, and 1.0% at 7.5, 37.5 and 87.5 micrograms/mL, respectively, as before. Analysis accuracy was assessed by spiking actual neutralization samples with IMPA or PMPA. For IMPA, the seven spike levels used ranged from 20 to 220% of the IMPA background level, and the incremental change in the found IMPA level ranged from 86 to 99 % of the true spiking increment (R2 = 0.9987 for the linear regression). For PMPA, the five spike levels ranged from 10 to 150% of the matrix background level, and similarly, the accuracy obtained ranged from 95 to 97% of the true incremental value (R2 = 0.9999 for the linear regression). PMID:9737210

Nassar, A E; Lucas, S V; Myler, C A; Jones, W R; Campisano, M; Hoffland, L D



Characterization of lymphoid cells by two-dimensional mini gel electrophoresis of proteins  

Microsoft Academic Search

Cytosol proteins from normal lymphocytes, leukemic lymphocytes, and different cultured lymphoid cell lines were separated by two-dimensional mini gel electrophoresis. By staining with Coomassie blue, specific protein patterns were obtained. Very similar gel maps were producted by the cytosol proteins of chronic lymphocytic leukemia cells, hairy cells, and of in vitro grown B cells. Protein 36\\/6.2 (molecular weight\\/isoelectric point), consistently

F. W. Hirsch; C. Bröckl; K. J. Bross; G. Dölken



A review of the CLIP system for the quantitative analysis of two-dimensional electrophoresis gels.  


This paper reviews the CLIP image processing system for the complete analysis of two-dimensional electrophoresis images. The analysis problem for two-dimensional gel images can be broken down into three issues: segmentation of individual gel images, alignment and comparison of pairs of gel images, and information storage and retrieval. This paper describes these problems and reviews how the CLIP system handles each of them. Segmentation is the location and isolation of each protein spot on an individual gel image and also the extraction of individual spot data such as position, area and volume. There are three basic stages: background field correction, noise filtering, spot detection and information extraction. Alignment and comparison of gel images involves matching protein spots between two gels. This can be quite difficult because there is not a simple relationship which can transform one gel image onto another. The database issues concern storing all the information which has been obtained from the above operations such that retrieval of this information can be readily performed. The advantage of the CLIP system over others is speed of processing. CLIP series computers use one processor for every pixel of the camera image such that image processing algorithms run in parallel. The main disadvantage is in the cost of these machines. With the declining trend in the cost of parallel processors, these machines will become more and more viable alternatives. This papers reviews the algorithms for the analysis of two-dimensional gels. It is shown that CLIP is flexible enough to perform more than one type of algorithm for a particular operation. PMID:2364927

Potter, D J



Multi-Channel Gel Electrophoresis and Continuous Fraction Collection Apparatus for High Throughput Protein Separation and Characterization.  

National Technical Information Service (NTIS)

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis col...

J. Jin M. Choi M. D. Biggin R. A. Nordmeyer



Development of a capillary electrophoresis platform for identifying inhibitors of protein-protein interactions.  


Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 are separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound-to-free ratio. The method was used to screen a library of 3443 compounds, and the results were compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that were reconfirmed in subsequent testing, suggesting greater specificity. This finding was attributed to the use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens, but at the current stage of development it is attractive as a secondary screen to test hits found by higher-throughput methods. PMID:24060167

Rauch, Jennifer N; Nie, Jing; Buchholz, Tonia J; Gestwicki, Jason E; Kennedy, Robert T



Recovery of protein by coomassie brilliant blue precipitation prior to electrophoresis.  


The interaction of protein with Coomassie Brilliant Blue G-250 results in formation of an insoluble protein-dye complex which can be recovered by centrifugation and redissolved for electrophoretic analysis. The precipitated protein can be washed in acetone to remove excess dye in order to enhance resolution. The residual dye becomes dissociated from the proteins on electrophoresis and can be exploited as a "dye front". The method allows simultaneous protein assay and recovery of microgram amounts of protein from dilute solution and could be widely applied for conserving, concentrating and desalting minute amounts of valuable sample prior to electrophoretic analysis. PMID:1282883

Marshall, T; Williams, K M



Quantitation of protein carbonylation by dot blot.  


Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is frequently measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent, 2,4-dinitrophenylhydrazine. We developed an immunochemical dot blot method for quantitation of protein carbonylation in homogenates or purified proteins. Dimethyl sulfoxide was employed as the solvent because it very efficiently extracts proteins from tissues and keeps them soluble. It also readily dissolves 2,4-dinitrophenylhydrazine and wets polyvinylidene difluoride (PVDF) membranes. The detection limit is 0.19 ± 0.04 pmol of carbonyl, and 60 ng of protein is sufficient to measure protein carbonyl content. This level of sensitivity allowed measurement of protein carbonylation in individual Drosophila. PMID:22326366

Wehr, Nancy B; Levine, Rodney L



Capillary electrophoresis of proteins in a quality control environment.  


A method for determining the purity of recombinant carboxypeptidase B utilizing CE-SDS has been developed and validated for use in a manufacturing quality control laboratory. The method was optimized, prior to validation, to allow for the lowest limit of quantitation. The method was validated with the typical ICHQ2A parameters, including accuracy, linearity, LOQ, precision, robustness/ruggedness, and specificity. All validation parameters met the acceptance criteria defined in the validation protocol. PMID:15163855

Good, David L; Cummins-Bitz, Stacey; Fields, Raeann M; Nunnally, Brian K



Introducing Proteomics in the Undergraduate Curriculum: A Simple 2D Gel Electrophoresis Exercise with Serum Proteins  

ERIC Educational Resources Information Center

Two-dimensional gel electrophoresis (2DGE) remains an important tool in the study of biological systems by proteomics. While the use of 2DGE is commonplace in research publications, there are few instructional laboratories that address the use of 2DGE for analyzing complex protein samples. One reason for this lack is the fact that the preparation…

Kim, Thomas D.; Craig, Paul A.



Introducing Proteomics in the Undergraduate Curriculum: A Simple 2D Gel Electrophoresis Exercise with Serum Proteins  

ERIC Educational Resources Information Center

|Two-dimensional gel electrophoresis (2DGE) remains an important tool in the study of biological systems by proteomics. While the use of 2DGE is commonplace in research publications, there are few instructional laboratories that address the use of 2DGE for analyzing complex protein samples. One reason for this lack is the fact that the preparation…

Kim, Thomas D.; Craig, Paul A.



Seed protein electrophoresis of wild and cultivated species of Celosia (Amaranthaceae)  

Microsoft Academic Search

Total seed storage proteins of five species of Celosia (14 taxa) have been compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Relative similarities between the taxa were estimated by Jaccard's similarity index and cluster analysis was performed to produce a UPGMA dendrogram which divides 14 taxa into two groups while C. trigyna holds an isolated position. One group includes 4x

P. Nath; D. Ohri; S. S. Jha; M. Pal



Proteomics: quantitative and physical mapping of cellular proteins  

Microsoft Academic Search

Genome sequencing provides a wealth of information on predicted gene products (mostly proteins), but the majority of these have no known function. Two-dimensional gel electrophoresis and mass spectrometry have, coupled with searches in protein and EST databases, transformed the protein-identification process. The proteome is the expressed protein complement of a genome and proteomics is functional genomics at the protein level.

Walter P Blackstock; Malcolm P Weir



Increase in local protein concentration by field-inversion gel electrophoresis  

PubMed Central

Background Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE). Results Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. Conclusion The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

Tsai, Henghang; Low, Teck Yew; Freeby, Steve; Paulus, Aran; Ramnarayanan, Kalpana; Cheng, Chung-pui Paul; Leung, Hon-chiu Eastwood



Quantitative determination of oxidized carbon nanotube probes in yeast by capillary electrophoresis with laser-induced fluorescence detection.  


Short oxidized multi-walled carbon nanotubes were functionalized with fluorescein isothiocyanate to form carbon nanotube probes (CNTP). The distribution of CNTP in yeast was quantitatively determined by capillary electrophoresis coupled with laser-induced fluorescence detection. The detection sensitivity for CNTP was greatly improved comparing with UV absorbance and Raman detection. The time- and temperature-dependent influx patterns of CNTP into yeast were obtained. The apparent permeability coefficient for influx of CNTP into yeast was calculated, which suggested that CNTP might permeate into yeast through endocytosis. This study implies that CNTP could be a fine drug transporter and might be wildly used in multidrug resistance research and microorganism detection. PMID:17723773

Xiao, Hua; Zou, Hanfa; Pan, Chensong; Jiang, Xiaogang; Le, X Chris; Yang, Ling



[Quantitative determinations of glycyrrhizic acid, glycyrrhetinic acid, morphine and sodium benzoate in compound liquorice tablets by capillary zone electrophoresis].  


Capillary zone electrophoresis (CZE) was used to quantitatively determine the contents of glycyrrhizic acid, glycyrrhetinic acid, morphine and sodium benzoate in compound liquorice tablets. The detection wavelength was 228 nm and the applied voltage was 14 kV. Borax solution of 50 mmol/L was used as the background electrolyte and hydrochlorothiazide as the internal standard. There were all good linear relationships between the concentrations and the relative areas of glycyrrhizic acid, glycyrrhetinic acid, morphine and sodium benzoate. Their average recoveries were 98.2%, 97.3%, 97.1% and 97.5%, respectively. The method is simple, rapid and accurate. PMID:12541626

Sun, Guo-xiang; Wang, Yu; Sun, Yu-qing



Two-dimensional gel electrophoresis revealed antipsychotic drugs induced protein expression modulations in C6 glioma cells.  


The efficacy and side effects of long-term administration of antipsychotic drugs (APDs) may be attributed to drug-induced change in protein expression in brain cells. Glial cells are non-neuronal cells that can provide nutrients and physiological support to neuronal cells. Glial cells are believed to participate in neurotransmission, neurons' early development, and guiding migration of neurons. Accumulated clinical data also indicate relationships between disturbance of glial cells' function and various psychotic diseases including schizophrenia. We used two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF mass spectrometry protein identification to analyze differentially expressed proteins in haloperidol-, risperidone-, and clozapine-treated C6 glioma cells. We found that the expression of pericentrin, glial fibrillary acidic protein, Rho GDP-dissociation inhibitor 1, anionic trypsin-1, peroxiredoxin-1, and parvalbumin were regulated by each of the three APDs. Western blot analysis supported the findings. Real-time quantitative PCR detected changed transcriptions of those proteins. Protein and gene expression of N-cadherin in C6 cells were affected by haloperidol and clozapine but not risperidone. In addition, regulatory effects of clozapine on the glyceraldehyde 3-phosphate dehydrogenase gene were observed in C6 cells. This may be the first study to uncover how APD-modulated genes may cause protein expression changes and affect ARHGDIA-mediated regulation of Rho GTPase family proteins in glial cells. PMID:22960606

Chen, Mao-Liang



Noncovalent labeling of proteins in capillary electrophoresis with laser-induced fluorescence detection  

Microsoft Academic Search

Interest in the use of capillary electrophoresis (CE) as a tool for protein separations continues to grow. Additionally, laser-induced\\u000a fluorescence (LIF) detection schemes promise ultrasensitive detection of small quantities of these important biomolecules\\u000a following their separation. In most cases, LIF detection of proteins necessitates their prior derivatization with a fluorescent\\u000a label molecule. To minimize the amount of additional sample handling

Christa Colyer



A general method for two-dimensional protein electrophoresis of fruit samples  

Microsoft Academic Search

During experiments characterizing and identifying proteins from controlled atmosphere-stored apple and peach fruit, we optimized methods for the extraction and two-dimensional electrophoresis (2-DE) of fruit proteins, using commercially available immobilized pH gradient strips for the first dimension. The method is relatively rapid with minimal handling of small amounts of sample, and has been reproduced successfully for 2-DE of a variety

Diane Barraclough; David Obenland; William Laing; Tanya Carroll



Plant protein isolation and stabilization for enhanced resolution of two-dimensional polyacrylamide gel electrophoresis  

Microsoft Academic Search

Two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) is the common method of choice for proteomic analysis. By introducing several small changes, a method was developed that not only improved the resolution and reproducibility of 2D–PAGE but also shortened the time of analysis. Precipitation by alkaline phenol and methanol\\/ammonium acetate was the choice for protein extraction. However, instead of precipitating the proteins overnight

Annamraju D. Sarma; Nathan W. Oehrle; David W. Emerich



Tear protein G originates from denatured tear specific prealbumin as revealed by two-dimensional electrophoresis.  


One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS PAGE) of the non-denatured low molecular weight (MW) tear proteins (dilution in phosphate buffered saline or in the non-ionic detergent Triton X100) revealed no protein G but a strongly marked 23-kD related to a tear specific prealbumin (TSP) subunit coming with the known 15, 17, 18 and 20-kD TSP subunits. Under mild denaturating conditions of sample preparation with SDS dilution just before electrophoresis, 23-kD protein decreased and a faint 32-kD protein G appeared. Under stronger denaturing conditions of sample preparation with SDS treatment (boiling or freeze-thaw cycles), 23-kD protein disappeared and two main protein G forms (32 and 34-kD) and additional bands (29, 36, 39, 42, 57 and 60-kD) appeared depending on the sample treatment. The isoelectric pH (pI) of these proteins ranged from pH 5.2 to pH 5.4. Different two-dimensional electrophoresis methods revealed that: - in presence of SDS, 23-kD protein was spontaneously changed into 17-kD TSP and such a phenomenon was partially reversible by using a non-ionic detergent (Triton X100), new proteins appeared under denaturating processes were related to various protein G forms and originated from TSP group, proteins G were produced by the aggregation of TSP subunit related to MW 17-kD/pI 5.0 corresponding to the major TSP subunit, disulfide bond formation was shown to play a major role in the aggregation process although protein G group was not totally reduced by dithiothreitol. Such results suggest that protein G is an in vitro experimental artifact due to denaturing conditions with SDS treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1483336

Baguet, J; Claudon-Eyl, V; Gachon, A M



Attomole Quantitation of Protein Separations with Accelerator Mass Spectrometry.  

National Technical Information Service (NTIS)

Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also preval...

J. S. Vogel P. G. Grant B. A. Buchholz K. Dingley K. W. Turtletaub



A protocol for protein extraction from lipid-rich plant tissues suitable for electrophoresis.  


Plant tissues contain high levels of nonprotein contaminants such as lipids, phenolic compounds, and polysaccharides among others, which interfere with protein extraction and electrophoretic separation. Preparation of good-quality protein extracts is a critical issue for successful electrophoretic analysis. Here, we describe a three-step method for protein extraction from lipid-rich plant tissues, which is suitable for both 1-D and 2-D electrophoresis and is compatible with downstream applications. The protocol includes prefractionation, filtration, and TCA/acetone precipitation steps prior to protein resolubilization. PMID:24136516

Zienkiewicz, Agnieszka; Rejón, Juan David; de Dios Alché, Juan; Rodríguez-García, María Isabel; Castro, Antonio Jesús



Quantitation of electrophoretically separated proteins in the submicrogram range by dye elution.  


A new method for the fast, reliable and reproducible submicrogram quantitation of proteins separated by different electrophoretic techniques is presented. The method is based on a modified sensitive staining technique using Coomassie Brilliant Blue G-250 in colloidal solution combined with an optimized elution procedure of the bound dye in a 3% w/v solution of sodium dodecyl sulfate followed by photometric determination of dye concentration in the eluate. In addition a new method is provided for background correction, even suitable for gels showing strong background staining. The staining procedure allows the detection of 20 ng depending on the nature of the protein and the separation technique used. Quantitation is linear at least in the range from 50 ng to 10 micrograms and highly reproducible even under non-optimized conditions. The presented method can be applied to sodium dodecyl sulfate-electrophoresis, isoelectric focusing and two-dimensional electrophoresis. PMID:7529171

Neumann, U; Khalaf, H; Rimpler, M



Three automated quantitative assays for serum proteins.  


Three quantitative assays for detection of proteins are reported. One is an adaptation to the Auto-Analyzer of the widely used method of Lowry et al. In another procedure, the product of the reaction of proteins with a modified biuret reacts with the phosphomolybdic--phosphotungstic reagent of Folin and Ciocalteu. The third method, performed on hydrolyzed sample, is based on the measurement of proline (Pro) and the conversion of the Pro content into protein. The universal presence of proline in serum proteins suggested this assay. The values for total serum proteins as assayed by the three procedures are similar. The second and the third assay for proteins, described in this paper, can also be performed manually. Studies on interfering substances and their elimination as well as on the sensitivity of the assays are reported. PMID:7363455

Blumenkrantz, N



Separation and relative quantitation of mouse plasma esterases with disc electrophoresis  

Microsoft Academic Search

A technique for the qualitative and quantitative study of mouse plasma esterases using disc elect rophoresis in acrylamide gel is described. This technique has been employed tK) study sub- strate specificity and quantitative relationships among various mouse strain and sex dependent esterases. Properties of acrylamide gels are discussed relative to their effect on the quaiitita- tive estimation of plasma esterases.




Precipitation of Champagne Base Wine Proteins Prior to 2D Electrophoresis.  


Numerous methods have been employed to depict the protein content of wines. Among them, two-dimensional electrophoresis (2D-E) presents a powerful resolution, but has been poorly applied to wine. Furthermore, 2D-E was coupled with various extraction methods of proteins without any reference method for wine. Here, we describe a rapid method to extract proteins from a champagne base wine through ultrafiltration followed by precipitation with ethanol and trichloroacetic acid. More than 50 spots were visualized on 2D-gels (7 cm, pH 3-6) by colloidal Coomassie Brilliant Blue staining. PMID:24136561

Cilindre, Clara



Sodium dodecyl sulfate-agarose gel electrophoresis of urinary proteins: application to multiple myeloma  

Microsoft Academic Search

We evaluated a new sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) for urinary protein analysis in patients with multiple myeloma (MM; n 5 47; ages, 62 6 2 years, mean 6 SE). Abnormal proteinuria (mean 5 1872 6 360 mg\\/24 h) was present in 95% of the samples; 75% of the patients had some sign of renal dysfunction (glomerular and\\/or tubular)

Thierry Le Bricon; Danielle Erlich; Djaouida Bengoufa; Michelle Dussaucy; Jean-Pierre Garnier; Bernard Bousquet


Sodium Dodecyl Sulfate-Polyacrylamide Gel Protein Electrophoresis of Freshwater Photosynthetic Sulfur Bacteria  

Microsoft Academic Search

Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis (SDS-PAGE) was carried out using different bacterial strains\\u000a of the photosynthetic sulfur bacteria Chlorobium, Thiocapsa, Thiocystis, and Chromatium cultured in the laboratory, and the natural blooms in two karstic lakes (Lake Cisó and Lake Vilar, NE Spain) where planktonic\\u000a photosynthetic bacteria (purple and green sulfur bacteria) massively developed accounting for most of the microbial

M. Begoña Osuna; Emilio O. Casamayor



Pulsed UV Laser-Induced Fluorescence Detection of Native Peptides and Proteins in Capillary Electrophoresis  

Microsoft Academic Search

A pulsed UV laser operating at 248 nm was used for the laser-induced fluorescence (LIF) detection of native tryptophan-containing compounds in capillary electrophoresis. The limit of detection (LOD) of tryptophan was found to be 3.3×10 M (S\\/N=2). The LODs of the model proteins conalbumin and bovine serum albumin were found to be 1.3×10 and 4×10 M (S\\/N=2), respectively. These results

King C. Chan; George M. Janini; Gary M. Muschik; Haleem J. Issaq



Characterization of discontinuous buffer junctions using pH indicators in capillary electrophoresis for protein preconcentration  

Microsoft Academic Search

An effective sample preconcentration technique for proteins and peptides was recently developed using capillary electrophoresis (CE) with discontinuous buffers [C.A. Nesbitt, J.T.-M. Lo, K.K.-C. Yeung, J. Chromatogr. A 1073 (2005) 175]. Two buffers of different pH created a junction to trap the sample molecules at their isoelectric points and resulted in over 1000-fold preconcentration for myoglobin within 30min. To study

Kristina Jurcic; Chandra A. Nesbitt; Ken K.-C. Yeung



Qualitative and quantitative assessment of marketed erythropoiesis-stimulating agents by capillary electrophoresis.  


Formulated erythropoiesis stimulating agents (ESAs) containing erythropoietin (EPO)-alpha, EPO-beta or darbepoetin-alpha were analyzed by capillary electrophoresis with a previously published method requiring no sample pre-treatment [1]. In this study, the method proved to be applicable to all formulations encountered, that is, in the presence of polysorbate 80, polysorbate 20 or human serum albumin as major excipients, thus extending the range of products that can be analyzed without pre-treatment. Method performance was evaluated and showed good linearity, range, precision and sensitivity. No significant matrix effects were observed for the various formulations. The ability of the method to resolve isoforms of each of the three active ingredients enabled comparison of the isoform distribution of finished products with that of the respective drug substance. In general, finished products and their corresponding drug substances showed similar isoform distribution and all were within manufacturer specifications. In addition, the content in active ingredient in the various dosage strengths was found to be in close agreement with the label claims with the exception of 2 out of 131 containers analyzed. Overall, this study demonstrated that the capillary zone electrophoresis method could be successfully applied to the analysis of most of the ESA products currently on the market in North America and Europe and that all products were found to have good batch-to-batch consistency. PMID:22954449

Boucher, Sylvie; Kane, Anita; Girard, Michel



Two-dimensional electrophoresis analysis of proteins extracted from Alexandrium sp. LC3  

NASA Astrophysics Data System (ADS)

Two-dimensional electrophoresis(2-DE) of protein extracted and purified from Alexandrium sp. LC3 was conducted. In the SDS-PAGE study, the relative molecular weights of the proteins were mainly in the range of 14kDa-31kDa and 43kDa-66kDa, and more proteins were detected between 14kDa and 31kDa. With the improved protein preparation, the two-dimensional electrophoresis patterns indicated that the relative molecular weights of the proteins were between 14kDa and 100kDa, and most of them ranged from 14kDa to 31kDa. This was consistent with the result of the SDS-PAGE analysis. The isoelectric points were found to lie between 3.0 and 8.0, and most of them were in the range of 3.0 6.0. Better separation effect was acquired with pre-prepared immobilized gradient (IPG) strip (pH3 5.6), and about 320 protein spots could be visualized on the 2-DE map by staining. Within pH3 10 and pH3 5.6 strips, the protein samples of Alexandrium sp. LC3 could be separated well.

Li, Hao; Miao, Jinlai; Cui, Fengxia; Li, Guangyou



Quantitative fluorescence spectral analysis of protein denaturation.  


This chapter describes a procedure of global analysis of the steady-state spectra measured with different concentrations of the denaturant to quantitatively study protein denaturation. With the help of physicochemical models, relevant spectral parameters that characterize the folding intermediate and thermodynamic parameters that describe a three-state model N?[Formula: see text]?I?[Formula: see text]?U can be estimated. PMID:24108622

van Stokkum, Ivo H M; Laptenok, Sergey P



Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry  

PubMed Central

Single-cell mass spectrometry (MS) empowers metabolomic investigations by decreasing analytical dimensions to the size of individual cells and subcellular structures. We describe a protocol for investigating and quantifying metabolites in individual isolated neurons using single-cell capillary electrophoresis hyphenated to electrospray ionization time-of-flight MS. The protocol requires ~2 h for sample preparation, neuron isolation, and metabolite extraction, and 1 h for metabolic measurement. The approach was used to detect more than 300 distinct compounds in the mass range of typical metabolites in various individual neurons (25–500-µm in diameter) isolated from the sea slug (Aplysia californica) central and rat (Rattus norvegicus) peripheral nervous systems. A subset of identified compounds was sufficient to reveal metabolic differences among freshly isolated neurons of different types and changes in the metabolite profiles of cultured neurons. The protocol can be applied to the characterization of the metabolome in a variety of smaller cells and/or subcellular domains.

Nemes, Peter; Rubakhin, Stanislav S.; Aerts, Jordan T.; Sweedler, Jonathan V.



Quantitative affinity electrophoresis of RNA-small molecule interactions by cross-linking the ligand to acrylamide.  


We show that the affinity electrophoresis analysis of RNA-small molecule interactions can be made quantifiable by cross-linking the ligand to the gel matrix. Using an RNA-aminoglycoside model system to verify our method, we attached an acryloyl chloride molecule to the aminoglycosides paromomycin and neomycin B to synthesize an acrylamide-aminoglycoside monomer. This molecule was then used as a component in gel polymerization for affinity electrophoresis, covalently attaching an aminoglycoside molecule to the gel matrix. To test RNA binding to the cross-linked aminoglycosides, we used the aminoglycoside binding RNA molecule derived from thymidylate synthase messenger RNA (mRNA) that contains a C-C mismatch. Binding is indicated by the difference in RNA mobility between gels with cross-linked ligand, with ligand embedded during polymerization, and with no ligand present. Critically, the predicted straight line relationship between the reciprocal of the relative migration of the RNA and the ligand concentration is obtained when using cross-linked aminoglycosides, whereas a straight line is not obtained using embedded aminoglycosides. Average apparent dissociation constants are determined from the slope of the line from these plots. This method allows an easy quantitative comparison between different nucleic acid molecules for a small molecule ligand. PMID:23928050

Boodram, Sherry N; McCann, Lucas C; Organ, Michael G; Johnson, Philip E



Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis of freshwater photosynthetic sulfur bacteria.  


Sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis (SDS-PAGE) was carried out using different bacterial strains of the photosynthetic sulfur bacteria Chlorobium, Thiocapsa, Thiocystis, and Chromatium cultured in the laboratory, and the natural blooms in two karstic lakes (Lake Cisó and Lake Vilar, NE Spain) where planktonic photosynthetic bacteria (purple and green sulfur bacteria) massively developed accounting for most of the microbial biomass. Several extraction, solubilization, and electrophoresis methods were tested to develop an optimal protocol for the best resolution of the SDS-PAGE. Protein composition from different water depths and at different times of the year was visualized within a molecular mass range between 100 and 15 kDa yielding up to 20 different protein bands. Protein banding patterns were reproducible and changed in time and with depth in agreement with changes in photosynthetic bacteria composition. When a taxonomically stable community was followed in time, differences were observed in the intensity but not in the composition of the SDS-PAGE banding pattern. Three environmental variables directly related to the activity of sulfur bacteria (light, oxygen, and sulfide concentrations) had a significant effect on protein banding patterns and explained 33% of the variance. Changes in natural protein profiles of the bacterial blooms agreed with changes in species composition and in the in situ metabolic state of the populations. PMID:20524118

Osuna, M Begoña; Casamayor, Emilio O



Study of Various Treatments to Isolate Low Levels of Cider Proteins to Be Analyzed by Capillary Sieving Electrophoresis  

Microsoft Academic Search

Four different sample treatments (ethanol precipitation, dialysis, ultrafiltration, and gel filtration) to isolate cider proteins were tested in this work. Purified cider proteins were analyzed by capillary sieving electrophoresis (CSE) using linear polyacrylamide as a sieving medium under optimized conditions; with this technique, separation, and molecular weight determination of proteins are possible. The electropherograms obtained in the protein analysis with

Domingo Blanco; Sara Junco; Yoana Expósito; Dolores Gutiérrez



Routine diagnosis with PhastSystem compared to conventional electrophoresis: automated sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining and western blotting of urinary proteins.  


The recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient-gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional "macro"-method with self-cast SDS-polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 microL sample volumes and an 8-25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS-PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha-1-antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS-PAGE and staining allows easy standardization of urine SDS-PAGE among clinical routine laboratories. PMID:2469571

Scherberich, J E; Fischer, P; Bigalke, A; Stangl, P; Wolf, G B; Haimerl, M; Schoeppe, W



[Improvement of two-dimensional gel electrophoresis of total proteins from rice anthers].  


This paper reported an improvement in 2-D gel electrophoresis of the proteome in Honglian cytoplasmic male sterile rice. An IPGphor unit with immobile pH gradient strips was used as the first dimension and SDS-PAGE as the second. The total anther proteins were extracted using TCA/acetone and then were washed 5-6 times with acetone till the proteins were white and clean, and then tributylphosphine and DTT were added into the rehydration buffer to improve the solubility of the proteins. The 2-D gel was stained by both methods of coomassie blue G-250 and silver. Extraction of proteins, pH of the strips and rehydration of the strips were optimized and compared. Higher repeatability and better separating protein pattern could be gained by this technique. PMID:17117559

Wen, Li; Liu, Gai; Wang, Kun; Peng, Xiao Jue; Wan, Cui Xiang; Li, Guo Min; Tao, Jun; Zhu, Ying Guo



Proteins isolated with TRIzol are compatible with two-dimensional electrophoresis and mass spectrometry analyses.  


TRIzol is used for RNA isolation but also permits protein recovery. We investigated whether proteins prepared with TRIzol were suitable for two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization mass spectrometry. Proteins from TRIzol-treated SH-SY5Y cells produced 2-DE spot patterns similar to those from an equivalent untreated sample. Subsequent identification of TRIzol-treated proteins using peptide mass fingerprinting was successful. TRIzol exposure altered neither the mass of myoglobin extracted from sodium dodecyl sulfate (SDS) gels nor the masses of myoglobin peptides produced by in-gel trypsin digestion. These findings suggest that proteins isolated with TRIzol remain amenable to proteomic analyses. PMID:22107888

Young, Clifford; Truman, Penelope



Proteomic Profiling Of Two-Dimensional Gel Electrophoresis Protein Expression Data  

NASA Astrophysics Data System (ADS)

We have undertaken two-dimensional gel electrophoresis (2-DE) proteomic profiling on a series of cell lines with different recombinant antibody production rates. Due to the nature of 2-DE proteomic investigations there will always be `process variability' factors in any data set collected in this way. Some of this variation will arise during sample preparation, gel running and staining, while further variation will arise from the gel analysis procedure. Therefore, in order to identify all significant changes in protein expression between biological samples when analysed by 2-DE, the system precision or `error', and how this correlates to protein abundance, must be known. Only then can the system be considered robust and investigators accurately and confidently report all observable statistically significant changes in protein expression. We introduce an expression variability test to identify protein spots whose expression correlates with increased antibody production. The results have highlighted a small number of candidate proteins for further investigation.

Ahmad, Norhaiza; Zhang, J.; Brown, P. J.; James, D. C.; Birch, J. R.; Racher, A. J.; Smales, C. M.



Evaluation of three-dimensional gel electrophoresis to improve quantitative profiling of complex proteomes.  


Two-dimensional remains one of the main experimental approaches in proteome analysis. However, comigration of protein leads to several limitations: lack of accuracy in protein identification, impaired comparative quantification, and PTM detection. We have optimized a third additional step of in-gel separation to alleviate comigration associated drawbacks. Spot resolution is strikingly improved following this simple and rapid method and the positive impact on protein and peptide identification from MS/MS data, on the analysis of relative changes in protein abundance, and on the detection of PTM is described. PMID:23592440

Colignon, Bertrand; Raes, Martine; Dieu, Marc; Delaive, Edouard; Mauro, Sergio



Quantitative proteomics: assessing the spectrum of in-gel protein detection methods  

PubMed Central

Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

Gauci, Victoria J.; Wright, Elise P.



Paper Electrophoresis of Saliva Albumins (Elektroforeza Bibulowa Bialek Sliny).  

National Technical Information Service (NTIS)

The proteins of mixed human saliva were concentrated by prevaporation, separated by electrophoresis and the fractions obtained there were of determined quantitatively. Three different techniques of separation were developed. The best separation was obtain...

T. Szymczyk



Suitability of staining techniques for the detection and quantitation of nonhistone high mobility group proteins.  


Three different staining techniques were compared for the detection of nonhistone high mobility group (HMG) proteins after acidic urea-polyacrylamide gel electrophoresis. Silver staining after glutaraldehyde fixation provides the highest detection sensitivity. Because of the acid solubility of HMG proteins special care has to be taken concerning fixation. Staining with colloidal CBB G-250 according to Neuhoff et al. is superior in sensitivity and reliability of quantitation when compared with noncolloidal Coomassie Brilliant Blue R-250. High detection sensitivity and reproducibility of quantitation are prerequisites for studying the tissue-specific expression of HMG proteins. In the present study tissue-specific differences in the molar amounts of various HMG proteins in thymus and erythrocytes of the chicken are documented by application of the methods tested. PMID:1692529

Csordas, A; Pedrini, M; Grunicke, H



Multidimensional high performance liquid chromatography--capillary electrophoresis separation of a protein digest: an update.  


The trypsin digest of a mixture of two proteins, namely cytochrome c and myoglobin, was first separated in the first dimension by high-performance liquid chromatography (HPLC). Fractions from the HPLC were collected every 30s with the aid of a fraction collector into a 96-well microtiter plate. After concentration, all the collected fractions were analyzed simultaneaosly in the second dimension by a 96-array capillary electrophoresis system. The labeled peptides were detected by laser-induced fluorescence. An internal standard, allura red, was added to all the fractions, prior to capillary electrophoretic analysis. The internal standard serves two functions, migration time correction and signal intensity correction. The data are presented in two different formats, as an electropherogram of all the fractions and in a two-dimensional (2-D) format. The 2-D plot of the data shows the density of each spot, which corresponds to the concentration of the migrating peptides. The total experimental time for the HPLC and capillary electrophoretic analyses ist less than 1 h, which ist much faster than using 2-D slab-gel electrophoresis or single-capillary capillary electrophoresis. PMID:11358138

Issaq, H J; Chan, K C; Liu, C S; Li, Q



A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis.  


Lightning Fast is a sensitive fluorescence-based stain for detecting proteins in one-dimensional and two-dimensional polyacrylamide electrophoresis gels. It contains the fluorophore epicocconone from the fungus Epicoccum nigrum that interacts noncovalently with sodium dodecyl sulfate and protein. Stained proteins can be excited optimally by near-ultraviolet light of about 395 nm or with visible light of about 520 nm. The stain can be excited using a range of sources used in image analysis systems including UVA (ca. 365 nm) and UVB (ca. 302 nm) transilluminators; Xenon-arc lamps; 488 nm and 457 nm Argon-ion lasers; 473 nm and 532 nm neodymium: yttrium aluminum garnet (Nd:YAG) solid-state lasers; 543 nm helium-neon lasers, and emerging violet, blue and green diode lasers. Maximum fluorescence emission of the dye is at approximately 610 nm. The limit of detection in one-dimensional gels stained with Lightning Fast protein gel stain is less than 100 pg of protein, rivaling the current limits of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Lightning Fast was found to be considerably more sensitive than SYPRO Ruby, SYPRO Orange, silver and Coomassie Brilliant Blue G-250 in matched experiments. Staining takes as little as 3.5 h and stained proteins displayed quantitative linearity over more than four orders of magnitude, thereby allowing visualization of entire proteomes. Lightning Fast protein gel staining is compatible with subsequent peptide mass fingerprinting using MALDI-MS and Edman-based sequencing chemistry. PMID:14673778

Mackintosh, James A; Choi, Hung-Yoon; Bae, Soo-Han; Veal, Duncan A; Bell, Philip J; Ferrari, Belinda C; Van Dyk, Derek D; Verrills, Nicole M; Paik, Young-Ki; Karuso, Peter



Assessing factors for reliable quantitative proteomics based on two-dimensional gel electrophoresis.  


We statistically analysed various factors to get accurate estimates of protein quantities from two-dimensional gels. Yeast proteins were labelled with (35)S or stained with Coomassie Brilliant Blue G-250, and spots were automatically quantified with software packages Kepler, ImageQuaNT, Melanie 3.0 and Progenesis. The different software packages proved to have very similar performances. With (35)S-labelled actin spot as a reference, we studied the staining efficiency of colloidal Coomassie blue as a function of amino acid composition of the protein, and derived an equation to estimate the number of molecules per cell from blue-stained proteins. Absolute quantification of most glycolytic enzymes was carried out in two yeast strains. PMID:15221754

Fiévet, Julie; Dillmann, Christine; Lagniel, Gilles; Davanture, Marlène; Negroni, Luc; Labarre, Jean; de Vienne, Dominique



Efficient solubilization buffers for two-dimensional gel electrophoresis of acidic and basic proteins extracted from wheat seeds  

Microsoft Academic Search

Plant tissues are made up of a broad range of proteins with a variety of properties. After extraction, solubilization of a diverse range of plant proteins for efficient proteomic analysis using two-dimensional electrophoresis is a challenging process. We tested the efficiency of 12 solubilization buffers in dissolving acidic and basic proteins extracted from mature seeds of wheat. The buffer containing

Gurusamy Chinnasamy; Christof Rampitsch



Quantitative determination of oxprenolol and timolol in urine by capillary zone electrophoresis.  


A simple capillary zone electrophoretic method with UV detection has been developed for the quantitative determination of the beta-adrenoreceptor antagonists (beta-blockers) oxprenolol and timolol in human urine, preceded by a solid-phase extraction step. The electrophoretic separation was performed on a 78 cm x 75 microm I.D. fused-silica capillary (effective capillary length: 70 cm). The electrolyte consisted of a Na2B4O7-H3BO3 (50 mM), pH 9. The introduction of the sample was made hydrostatically for 20 s and the running voltage 25 kV at the injector end of the capillary. Photometric detection was used at a wavelength of 229 nm for oxprenolol and 280 nm for timolol. Under these conditions oxprenolol migrated at 4.76+/-0.05 min and timolol at 4.97+/-0.05 min. The solid-phase extraction methods were optimised for each beta-blocker and provided recoveries of 72.8% for timolol and 94.52% for oxprenolol. Good resolution from the endogenous compounds present in the urine matrix were achieved for both compounds. The method was applied to the determination of both beta-blockers in pharmaceutical formulations and urine samples obtained from hypertensive patients after the ingestion of a therapeutic dose (in a 24-h time interval after the ingestion). The quantitative results were compared with results previously obtained at our laboratories by HPLC and were found to be in good agreement. Good reproducibility, linearity, accuracy and quantitation limits (in urine) of 0.19 microg/ml for timolol and 0.20 microg/ml for oxprenolol were obtained, allowing the method to be applied to pharmacokinetic studies of these compounds. PMID:11999762

Maguregui, M I; Jiménez, R M; Alonso, R M; Akesolo, U



Determination of eight genetically modified maize events by quantitative, multiplex PCR and fluorescence capillary gel electrophoresis  

Microsoft Academic Search

Specific legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should\\u000a be labelled. This has necessitated the development of methods for detection and quantification of such materials. Here we\\u000a present a robust, quantitative, 9-plex PCR method for event-specific detection of maize TC1507, MON863, MON810, T25, NK603,\\u000a GA21, construct specific detection of BT11, BT176

Bjarte R. Heide; Signe M. Drømtorp; Knut Rudi; Even Heir; Askild L. Holck



Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots  


After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

Zhang, Jian-Shi (Shanghai, CN); Giometti, Carol S. (Glenview, IL); Tollaksen, Sandra L. (Montgomery, IL)




Microsoft Academic Search

Protein electrophoresis, hematological and cholinesterase values were determined in 32 nestling free-living peregrine falcons (Falco peregrinus) (15- to 27-days-old) in order to estab- lish normal reference values for this population. The following values (mean SD) were ob- served: prealbumin 0.31 0.04 g\\/dl, albumin 1.25 0.06 g\\/dl, 1 and 2-globulin 0.23 0.02 and 0.16 0.02 g\\/dl respectively, -globulin 1.02 0.05 g\\/dl,

Maria del Pilar Lanzarot; Andres Montesinos; Manuel Ignacio San Andres; Casilda Rodrõ ´ guez; Marõ ´ a; Victoria Barahona


Two-dimensional electrophoresis of proteins in human serum: improved resolution by use of narrow pH gradients and prolonged electrophoresis.  


The limited resolution of serum proteins achieved with a simplified technique of two-dimensional electrophoresis (Clin. Chim. Acta 103: 51-59, 1980) has been improved by using different ampholyte (Ampholine) mixtures in the first dimension, to obtain relatively shallow pH gradients, and prolonged electrophoresis time in the second dimension. The technique has been further simplified, without negative effect, by decreasing the concentration of non-ionic detergent (first dimension), omitting both sodium dodecyl sulfate equilibration and the use of a stacking gel (second dimension), and by using an improved silver-staining procedure. Reverse-polarity isoelectric focusing and non-equilibrium pH-gradient electrophoresis have been successfully used to resolve the basic polypeptides in serum after deleting sodium dodecyl sulfate from the sample preparation. These combined techniques reveal over 1100 polypeptides in human serum. After immunodeletion of albumin, additional serum polypeptides are seen. Immunodeletion of serum proteins from plasma reveals polypeptides that are relatively specific to plasma. PMID:6499174

Marshall, T; Williams, K M; Vesterberg, O



Quantitative study on selective stacking of zwitterions in large-volume sample matrix by moving reaction boundary in capillary electrophoresis.  


The paper advanced the theoretical procedures for quantitative design on selective stacking of zwitterions in full capillary sample matrix by a cathodic-direction moving reaction boundary (MRB) in capillary electrophoresis (CE) under control of electroosmotic flow (EOF). With the procedures, we conducted the theoretical computations on the selective stacking of two test analytes of L-histidine (His) and L-tryptophan (Trp) by the MRB created with 30 mM pH 3.0 formic acid-NaOH buffer and 2-80 mM sodium formate. The results revealed the following three predictions. At first, the MRB cannot stack His and Trp plugs if less than 12.5 mM sodium formate is used to form the MRB and prepare the sample matrix. Second, the MRB can stack His and/or Trp sample plugs completely if higher than 50 mM sodium formate is chosen to form the MRB. Third, the MRB can only focus His plug completely, but stack Trp plug partially if 20-50 mM sodium formate is used; this implied the complete MRB-induced selective stacking to His rather than Trp. All the three predictions were quantitatively proved by the experiments. With great dilution of sample matrix and control of EOF, controllable, simultaneous and MRB-induced selective stacking and separation of zwitterions were achieved. The theoretical results hold evident significances to the quantitative design of selective stacking conditions and the increase of detection sensitivity of zwitterions in CE. In addition, the control of EOF by cetyltrimethylammonium bromide (CTAB) can evidently improve the stacking efficiency to both His and Trp. PMID:16041697

Qin, Wei-Hua; Cao, Cheng-Xi; Li, Shan; Zhang, Wei; Liu, Wei



Analysis of major milk whey proteins by immunoaffinity capillary electrophoresis coupled with MALDI-MS.  


Two major milk whey proteins, ?-lactoglobulin and ?-lactalbumin, are among the main cow milk allergens and can cause allergy even at a very low concentrations. Therefore, these proteins are interesting targets in food analysis, not only for food quality control but also for highlighting the presence of allergens. Herein, a sensitive analysis for ?-lactoglobulin and ?-lactalbumin was developed using immunoaffinity capillary electrophoresis hyphenated with MALDI-MS. Magnetic beads functionalized with appropriate antibodies were used for ?-lactoglobulin and ?-lactalbumin immunocapture inside the capillary. After elution from the beads, analyte focusing and separation were performed by transient isotachophoresis followed by MALDI-MS analysis performed through an automated iontophoretic fraction collection interface. A LOD in the low nanomolar range was attained for both whey proteins. The method developed was further applied to the analysis of different milk samples including fortified soy milk. PMID:22887160

Gasilova, Natalia; Gassner, Anne-Laure; Girault, Hubert H



Control of the Staining Procedure after Paper Electrophoresis  

Microsoft Academic Search

WHEN quantitative techniques are attempted for the analysis of serum proteins separated by electrophoresis on filter paper, it is recognized that the staining procedure, followed by removing the surplus stain, constitutes sources of error. In order to get more satisfactory reproducibility we apply on each paper strip (after electrophoresis but before staining) 0.02 ml. of a 0.05 per cent solution

Ch. Wunderly



Using affinity capillary electrophoresis to determine binding stoichiometries of protein-ligand interactions.  


We have developed a new method utilizing affinity capillary electrophoresis (ACE) for the determination of binding stoichiometries in biochemical systems. Using the same concentration of a ligand in the sample and the electrophoresis buffer, the appearance of an inverted peak corresponding to the free ligand in the resulting electropherogram provides a criterion of binding of a ligand to its receptor protein. For both low (fast off rates) and high (slow off rates) affinity systems, analysis of the integration of free ligand peak in electropherograms as a function of the total concentration of a ligand in samples at constant concentration of receptor protein yields the binding stoichiometry of the ligand to the protein. Applications of this technique to studies of (i) the inhibition of carbonic anhydrases (CA, EC, from human and bovine erythrocytes) by 4-alkylbenzenesulfonamide 1, (ii) the interaction of a monoclonal antibody to human serum albumin (anti-HSA) with its antigen HSA, and (iii) the binding of streptavidin (from Streptomyces avidinii) to biotin derivatives (monobiotinylated oligodeoxyribonucleotide 2, fluorescein biotin, or Lucifer Yellow biotin) yield stoichiometries of 1:1, 1:2, and 1:4, respectively. For multivalent, tight-binding systems, this ACE method can readily separate stable intermediate species. This method is generally applicable to both tight- and weak-binding systems, requires only nanograms of proteins and ligands, involves no radioactive materials, and does not require changes in electrophoretic mobilities of receptor proteins upon binding with ligands. It thereby provides a rapid, sensitive, and convenient method for measuring binding stoichiometries of ligands to proteins. PMID:8075061

Chu, Y H; Lees, W J; Stassinopoulos, A; Walsh, C T



Quantitative Immunofluorescent Blotting of the Multidrug Resistance-associated Protein 2 (MRP2)  

PubMed Central

Introduction Quantitation of the expression levels of proteins involved in drug transport and disposition is needed to overcome limitations of film-based detection of chemiluminescent immunoblots. Purpose The purpose was to describe and validate a quantitative immunofluorescent blotting method for detection of ATP-Binding Cassette Transporter Isoform C2/Multidrug Resistance-associated Protein 2 (ABCC2/MRP2). Methods Western blotting was performed by electrophoresis of membrane vesicle protein isolated from Sf9 cells overexpressing MRP2 subsequently blotting with infrared labeled secondary antibody. The bound complex was detected using the Odyssey Infrared Imaging System (Li-Cor; Lincoln, NE). The images were analyzed using the Odyssey Application Software to obtain the integrated intensities, followed by linear regression of the intensity data. Results The limits of quantitation for the time-insensitive technique described here were from 0.001?g to 0.5?g of total membrane protein, the coefficient of variation of the slope was 8.9%; r2 values were 0.986 ± 0.012. The utility and sensitivity of this technique was demonstrated in quantitating expression of MRP2 in human placental tissue samples, in which MRP2 was present in low abundance. Discussion The immunofluorescent blotting technique described provides sensitive, reproducible, and quantitative determinations of large, integral membrane proteins such as MRP2, all with potential long-term cost savings.

Gerk, Phillip M.



Two-dimensional gel electrophoresis and immunoblotting of Campylobacter pylori proteins.  

PubMed Central

Whole-cell, outer-membrane protein, flagellum-associated antigens and partially purified urease of Campylobacter pylori were analyzed by two-dimensional gel electrophoresis. C. pylori strains were readily distinguished from strains of Campylobacter jejuni, C. coli, and C. fetus by absence of major outer membrane proteins with Mrs of 41,000 to 45,000. C. pylori strains also lacked the acidic surface-array proteins at Mr 100,000 to 149,000 identified previously in serum-resistant strains of C. fetus. Surface labeling of intact C. pylori cells with 125I revealed two common major proteins, which we have designated protein 2 (pI 5.6 to 5.8, Mr 66,000) and protein 3 (pI 5.2 to 5.5, Mr 63,000). Proteins 2 and 3 were also the major components (subunits) observed in partially purified urease. Partially purified preparations of flagella consistently contained proteins 2 and 3. Thus, urease appears to be associated with both outer membranes and flagella of C. pylori. C. pylori strains also possessed an antigen at Mr 59,000 which was cross-reactive with antiserum against flagella of C. jejuni. However, the antigen did not appear to be associated with flagella per se in C. pylori. Protein 2 was unique to C. pylori among the Campylobacter species studied. It was not recognized by antibody against whole cells of C. jejuni or C. fetus or flagella of C. jejuni. Protein 3 was cross-reactive with antiserum against whole cells of C. jejuni and C. fetus, as were several other major protein antigens. Because protein 2 is a major outer membrane protein that is apparently unique to C. pylori, development of monospecific antibodies against this antigen may be useful for the identification of C. pylori in tissues, and purified antigen may be useful for serologic tests for specific diagnosis of C. pylori infections. Images

Dunn, B. E.; Perez-Perez, G. I.; Blaser, M. J.



Quantitation of proteins separated in N, N'-1,2-dihydroxyethylenebisacrylamide-crosslinked polyacrylamide gels.  


A simple and rapid method for the quantitation of proteins separated either by sodium dodecyl sulfate-electrophoresis or by isoelectric focusing in slab gels is presented. The method is based on the solubility of polyacrylamide gels crosslinked with N, N'-1, 2-dihydroxyethylenebisacrylamide (DHEBA) in periodic acid. After electrophoretic separation proteins are stained with Coomassie brilliant blue G-250. DHEBA gels show considerable swelling during the staining and destaining process but can be shrunk to their normal size in a 10% (w/v) solution of ammonium sulfate. Stained bands are cut from the gel and solubilized in periodic acid. During dissolution the dye decolorizes. Protein concentration in the solution is determined by a modified Coomassie dye-binding assay. Quantitation is linear in the range of 100 ng to 5 micrograms and not disturbed by dissolved gel. Separations in N, N'-1, 2-dihydroxyethylenebisacrylamide-crosslinked gels show qualities similar to those in normal crosslinked gels. PMID:1456419

Neumann, U; Khalaf, H; Rimpler, M



Differential Expression of Proteins in Lung Cancer Using Difference in Gel Electrophoresis (DIGE)  

PubMed Central

Background: Lung cancer remains the leading cause of cancer-related mortality worldwide. Early detection of lung cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. The purpose of this study was to develop techniques to identify the differential expression protein profiles between tumor and tumor free of lung cancer tissues. Methods: 2-dimensional differential ingel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to analyze four samples of lung cancer tissue (3 replicates each). Results: From optimized 2DE image, A total of 2561 spots were detected and 427 spots of these were differentially expressed (p<0.01). 40 spots were subjected to mass spectrometry including over expressed proteins and under expressed proteins. Some proteins were the products of oncogenes and others were involved in the regulation of cell cycle and signal transduction such as Annexin III, Selenium binding protein, glyceraldehydes-3-phosphate dehydrogenase, cathepsin D and catalase. Conclusion: These data are valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis, establishing human lung cancer proteome database and screening molecular marker to further study human lung cancer. Using the DIGE approach, we were able to find many proteins that were expressed differently due to the disease state (tumor and tumor-free).

Beckett, P.; Aulak, K.S.; Masri, F.



A plant dye from Lawsonia inermis for protein staining after polyacrylamide gel electrophoresis.  


A reddish-brown dye was isolated from the leaves of Lawsonia inermis by extraction with calcium hydroxide (pH 11-12). A 3.6% crude extract in ethanol/water, 1:1 v/v, was used for direct staining, without fixation, of bovine serum albumin, casein and human serum proteins, following polyacrylamide gel electrophoresis in cylindrical gels. After staining for 30 min the gels were destained for 1/2-2 h with 7% acetic acid at 60 degrees C. Protein staining with Amido Black 10B and Coomassie Brilliant Blue R-250, according to standard protocols, required destaining for 24 h and more to obtain a comparably cleared background. Staining with the plant dye and Coomassie Brilliant Blue had a similar overall staining sensitivity but some minor components of human serum showed different staining characteristics with each of the two dyes. Staining with the plant dye excels over standard staining by speed and simplicity. PMID:1692790

Ali, R; Sayeed, S A



Genetic diversity of cattle in south China as revealed by blood protein electrophoresis.  


Genetic variation of 31 blood protein loci in 236 cattle from eight South China populations (including mithan, Bos frontalis) and a Holstein population was investigated by means of horizontal starch gel electrophoresis. Thirteen loci (ALB, CAR, Hb-b, Np, PGM, Amy-I, PEP-B, AKP, 6PGD, Cp, Pa, EsD, and TF) were found to be polymorphic. The comparison of average heterozygosities (H) shows that all the native cattle embrace a rich genetic diversity. Our results on protein polymorphism suggest that cattle in China originated mainly from Bos indicus and Bos taurus; Xuwen, Hainan, Wenshan, and Dehong cattle and the Dehong zebu are close to zebu-type cattle, and Diqing and Zhaotong cattle are close to the taurine. The mithan was very different from other native cattle, and we suggest that its origin was complicated and may be influenced by other cattle species. PMID:10624516

Nie, L; Yu, Y; Zhang, X Q; Yang, G F; Wen, J K; Zhang, Y P



Suitability of two-dimensional electrophoretic protein separations for quantitative detection of mutations  

SciTech Connect

Separation of proteins by two-dimensional electrophoresis (2DE) provides a powerful method for mutagenesis studies, since hundreds of proteins can be monitored simultaneously. In previous mutation studies in which 2DE has been used, only qualitative protein differences were monitored; quantitative protein variations were not evaluated. Although significant differences in protein abundance can be detected by eye, the large number of protein spots present in 2DE patterns together with the large number of individual patterns required for a mutagenesis study would necessitate the use of a computerized analysis system to detect the rare quantitative protein changes indicative of gene deletions or inactivation of genes by point mutations in regulatory genes. A pilot study to search for heritable mutations induced by treatment of mice with either ethylnitrosourea or gamma radiation is underway. Samples are being monitored for quantitative changes that reduce the amount of protein by about 50%. The results of this study indicate that the key methods to improve the application of 2DE to mutation screening are to increase the number of measurable spots (i.e., improve stain sensitivity) and to decrease the spread of values for the volume measurements. Even small improvements in these areas could greatly increase the number of monitorable spots. 9 refs., 4 figs.

Taylor, J.; Anderson, N.L.; Anderson, N.G.; Gemmell, A.; Giometti, C.S.; Nance, S.L.; Tollaksen, S.L.



Quantitation of collagen types I, III and V in tissue slices by capillary electrophoresis after cyanogen bromide solubilization.  


A method for the determination of the proportions of major fiber-forming collagens (types I, III and V) in soft connective tissue was elaborated. The method is based on the release of insoluble collagen by CNBr with subsequent separation of the arising peptides. For routine application the peptides are separated by capillary electrophoresis (50 mM phosphate pH 2.5, 15 kV, 50 degrees C, 70/60 cm x 70 microns I.D. capillary with UV detection at 200 nm). Quantitation of collagen type I can be done either on the basis of spiking the sample with a peptide mixture obtained from a known amount of collagen type I, or by spiking the sample with an equimolar mixture of the two peptides [alpha 1(I)CB2 and alpha 1(I)CB4] (constituting a fused peak) along with alpha 1(III)CB2 and alpha 1(V)CB1. Compared to the previously published methods the procedure is faster and does not require isolation of marker peptides by tedious chromatographic procedures in a preceding preparatory step. Good results are obtained within a wide range of run buffer concentrations and applied voltages; conversely, intensive cleaning of the capillary after every three runs is recommended with a new capillary after 20-30 runs. PMID:9061493

Deyl, Z; Novotná, J; Miksík, I; Jelínková, D; Uhrová, M; Suchánek, M



Mobility-based selective on-line preconcentration of proteins in capillary electrophoresis by controlling electroosmotic flow  

Microsoft Academic Search

A simple method to perform selective on-line preconcentration of protein samples in capillary electrophoresis (CE) is described. The selectivity, based on protein electrophoretic mobility, was achieved by controlling electroosmotic flow (EOF). A short section of dialysis hollow fiber, serving as a porous joint, was connected between two lengths of fused silica capillary. High voltage was applied separately to each capillary,

Qinggang Wang; Bingfang Yue; Milton L. Lee



Membranes of the adrenal medulla. Behaviour of insoluble proteins of chromaffin granules on gel electrophoresis  

PubMed Central

Washed membranes of bovine adrenal chromaffin granules contained most of the cholesterol and phospholipids of the particle and 22% of the total protein. The protein/lipid ratio was about 0.45 (w/w). Dopamine(3,4-dihydroxyphenethylamine)?-hydroxylase, Mg2+-activated nucleoside triphosphatase and cytochrome b-559 activities were present in the membrane. ATP was the best substrate for the nucleoside triphosphatase, whose pH optimum was 6.4, Km 7×10?4m and Vmax. 1.8?mol/h per mg of protein. Treatment of the membranes with various detergents caused a preferential solubilization of protein compared with lipids. Membranes dissolved in sodium dodecyl sulphate or phenol–acetic acid–urea were subjected to polyacrylamide-gel electrophoresis at alkaline and acid pH respectively. The electrophoretic patterns given by the proteins of the chromaffin granule membrane were distinct from those given by the chromogranins, and from those given by mitochondrial and microsomal membrane proteins. ImagesPLATE 1

Winkler, H.; Hortnagl, Heide; Hortnagl, H.; Smith, A. D.



Protein and LDH-isoenzyme pattern of the urine from patients with diabetes mellitus determined by disc-electrophoresis  

Microsoft Academic Search

Summary  We have examined U-LDH excretion, protein excretion (a.m. Kjeldahl) and, by means of disc electrophoresis, the pattern of protein and LDH isoenzymes in urine from 73 diabetics. The diabetics are classified into JDM: 0–24 years, ADM: 25–64 years, EDM: > 65 years, Recent JDM: duration 1 year, nephropathy: protein excretion 500 mg\\/24h. — Conclusions: There is increased urinary protein excretion

L. Hemmingsen; N. Høiby; P. Kragh-Søbensen



Protein degradation in human pure pancreatic juice analyzed by two-dimensional gel electrophoresis.  


Two-dimensional gel electrophoresis (2-DE) was used to study protein degradation in human pure pancreatic juice (PPJ) which was collected at 5 min intervals for 20 min by selective endoscopic cannulation of the main pancreatic duct. In PPJ collected from healthy subjects no significant degradation was observed by incubating PPJ at 37 degrees C up to 6 h. By further incubation for 24 h, glycoprotein-1, procarboxypeptidase A-1 and lipase were nearly completely degraded, while alpha-amylase and procarboxypeptidase B-1 were not degraded under these conditions; alpha-amylase became labile in the presence of 1 mM ethylene diaminetetraacetic acid (EDTA) or 10 mM phenyl methyl sulfonyl fluoride (PMSF). Protein degradation was observed by 2-DE of an initial fraction of PPJ collected from patients with chronic calcific pancreatitis (CCP). The 2-DE patterns of subsequent fractions resembled those of PPJ from healthy subjects. The mixture of the last fraction with the initial fraction showed significant protein degradation, inhibited by adding aprotinin. Furthermore, the extent of protein degradation correlated with the dilatation of the main pancreatic duct as a consequence of intraductal stagnation of pancreatic juice. These findings demonstrate that protein degradation in PPJ is accelerated by intraductal activation of serine proteases in the case of patients with CCP. 2-DE of PPJ from patients with CCP provides useful information for the evaluation of intraductal activation of zymogens and the progress of chronic pancreatitis. PMID:8738347

Furui, T; Ikeda, M; Chao-Ming, L; Okita, K; Nakamura, K



High resolution two-dimensional gel electrophoresis of human erythrocyte membrane proteins.  

PubMed Central

Three different two-dimensional (2-D) gel electrophoretic techniques have been modified to provide high resolution of human erythrocyte membrane proteins. The resulting gels were referenced to the established one-dimensional (1-D) sodium dodecylsulfate (SDS) gel electrophoretic profile, and the effects of endogenous proteolysis and cytosolic contamination were studied. It is concluded that in vitro proteolysis and cytosolic contamination do not contribute significantly to the patterns observed on the 2-D gels, under the conditions used for erythrocyte ghost preparation. The procedures require only small quantities of blood; as many as twenty 2-D gel profiles can be obtained from 5 ml of blood. The combination of nonequilibrium isoelectric focusing (IEF) in the first dimension, SDS electrophoresis in the second dimension, and very sensitive silver staining techniques resolves more than 250 individual protein spots. This appears to be the most useful single procedure for the analysis of red cell membrane proteins. Membrane protein profiles from patients with Duchenne muscular dystrophy, Wernicke-Korsakoff syndrome, and acanthocytosis with degeneration of the basal ganglia were compared with normal controls. The patterns for Duchenne muscular dystrophy and Wernicke-Korsakoff syndrome were not different from normal patterns. The pattern for the patient with acanthocytosis and degeneration of the basal ganglia consistently showed a high level for one protein in the 100,000 mol. wt. range. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5

Copeland, B R; Todd, S A; Furlong, C E



Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots  


After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a dc power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. 8 figs.

Zhang, Jian-Shi; Giometti, C.S.; Tollaksen, S.L.



Folding/unfolding/refolding of proteins: present methodologies in comparison with capillary zone electrophoresis.  


A series of techniques for monitoring protein folding/unfolding/misfolding equilibria are here assessed and compared with capillary zone electrophoresis (CZE). They include spectroscopic techniques, such as circular dichroism, intrinsic fluorescence, nuclear magnetic resonance, Fourier transform infrared and Raman spectroscopy, small-angle X-ray scattering, as well as techniques based on biological assays, such as limited proteolysis and immunochemical analysis of different conformational states. Some unusual probes, such as mass spectrometry for probing unfolding transitions, are also discussed. Size-exclusion chromatography is also evaluated in view of the fact that this technique, like all electrophoretic techniques, and unlike spectroscopic probes, which can only see an average signal in mixed populations, can indeed physically separate folded vs. unfolded macromolecules, especially in the case of slow equilibria. Particular emphasis is devoted to electrophoretic techniques, such as gel-slab electrophoresis in transverse urea or thermal gradients, and CZE. In the latter case, a number of applications are shown, demonstrating the excellent correlation of CZE with more traditional probes, such as intrinsic fluorescence monitoring. It is additionally shown that CZE can be used for measuring the deltaG degrees of unfolding over the pH scale, in good agreement with theoretical calculations on the electrostatic free energy of folding vs. pH, as calculated with a linearized Poisson-Boltzmann equation. Finally, it is demonstrated that CZE can probe also aggregate formation in the presence of helix-inducing agents, such as trifluorethanol. PMID:11519938

Righetti, P G; Verzola, B



Identification of Campylobacter jejuni and C. coli by gel electrophoresis of the outer membrane proteins.  

PubMed Central

Analysis of the electrophoretic profiles of the outer membrane proteins could be used to differentiate Campylobacter jejuni (16 strains) from Campylobacter coli (10 strains). This observation was confirmed by the study of DNA homology obtained by a quantitative filter hybridization method. The hippurate hydrolysis test gave a poor correlation with the results of differentiation obtained by DNA homology studies and outer membrane protein profile. Images

Derclaye, I; Delor, I; Van Bouchaute, M; Moureau, P; Wauters, G; Cornelis, G R



In-gel staining of proteins in native poly acryl amide gel electrophoresis using tetrakis(4-sulfonato phenyl)porphyrin.  


Protein identification in polyacrylamide gel electrophoresis (PAGE) requires post-electrophoretic steps like fixing, staining and destaining of the gel, which are time-consuming and cumbersome. We have developed a method for direct visualization of protein bands in PAGE using tetrakis(4-sulfonato phenyl)porphyrin (TPPS) as a dye without the need for any post electrophoretic steps, where separation and recovery of enzymes become much easier for further analysis. Activity staining was done to prove that the biochemical activity of the enzymes was preserved after electrophoresis. PMID:21233569

Divakar, Kalivarathan; Sujatha, Vijayan; Barath, Sridhar; Srinath, Krishnamurthy; Gautam, Pennathur



In-gel staining of proteins in native polyacrylamide gel electrophoresis using meso-tetrakis(4-sulfonatophenyl) porphyrin.  


Protein identification in polyacrylamide gel electrophoresis (PAGE) requires post-electrophoretic steps like fixing, staining, and destaining of the gel, which are time-consuming and cumbersome. A new method for direct visualization of protein bands in PAGE has been developed using meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) as a dye without the need for any post-electrophoretic steps; thus, separation and recovery of enzymes become much easier for further analysis. Activity staining was carried out to show that the biochemical activity of the enzymes was preserved after electrophoresis. PMID:22585523

Divakar, K; Devi, G Nandhini; Gautam, Pennathur



Volatile kinetic capillary electrophoresis for studies of protein-small molecule interactions.  


Kinetic capillary electrophoresis (KCE) is a toolset of homogeneous affinity methods for studying kinetics of noncovalent binding. Sensitive KCE measurements are typically done with fluorescence detection and require a fluorescent label on a smaller-sized binding partner. KCE with fluorescence detection is difficult to use for study of protein-small molecule interactions since labeling small molecules is cumbersome and can affect binding. A combination of KCE with mass-spectrometry (KCE-MS) has been recently suggested for label-free studies of protein-small molecule interactions. The major obstacle for studies by KCE-MS is a buffer mismatch between KCE and MS; MS requires volatile buffers while KCE of protein-ligand interactions is always run in near-physiological buffers. Here we asked a simple question: can protein-ligand interactions be studied with KCE in a volatile buffer? We compared three volatile buffers (ammonium acetate, ammonium bicarbonate, and ammonium formate) with a near-physiological buffer (Tris-acetate) for three protein-ligand pairs. The volatile buffers were found not to significantly affect protein-ligand complex stability; moreover, when used as CE run buffers, they facilitated good-quality separation of free ligands from the protein-ligand complexes. The use of volatile buffers instead of Tris-acetate in detection of small molecules by MS improved the detection limit by approximately 2 orders of magnitude. These findings prove the principle of "volatile" KCE, which can be easily coupled with MS to facilitate label-free kinetic studies of protein-small molecule interactions. PMID:22823518

Bao, Jiayin; Krylov, Sergey N



Microchip capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins using uncoated Ormocomp microchips.  


We present rapid (<5 min) and efficient intact protein analysis by mass spectrometry (MS) using fully microfabricated and monolithically integrated capillary electrophoresis-electrospray ionization (CE-ESI) microchips. The microchips are fabricated fully of commercial inorganic-organic hybrid material, Ormocomp, by UV-embossing and adhesive Ormocomp-Ormocomp bonding (CE microchannels). A sheath-flow ESI interface is monolithically integrated with the UV-embossed separation channels by cutting a rectangular emitter tip in the end with a dicing saw. As a result, electrospray was produced from the corner of chip with good reproducibility between parallel tips (stability within 3.8-9.2% RSD). Thanks to its inherent biocompatibility and stable (negative) surface charge, Ormocomp microchips enable efficient intact protein analysis with up to ?10(4) theoretical separation plates per meter without any chemical or physical surface modification before analysis. The same microchip setup is also feasible for rapid peptide sequencing and mass fingerprinting and shows excellent migration time repeatability from run to run for both peptides (5.6-5.9% RSD, n=4) and intact proteins (1.3-7.5% RSD, n=3). Thus, the Ormocomp microchips provide a versatile new tool for MS-based proteomics. Particularly, the feasibility of the Ormocomp chips for rapid analysis of intact proteins with such a simple setup is a valuable increment to the current technology. PMID:22152798

Sikanen, Tiina; Aura, Susanna; Franssila, Sami; Kotiaho, Tapio; Kostiainen, Risto



Western blotting using microchip electrophoresis interfaced to a protein capture membrane.  


Western blotting is a commonly used assay for proteins. Despite the utility of the method, it is also characterized by long analysis times, manual operation, and lack of established miniaturized counterpart. We report a new way to Western blot that addresses these limitations. In the method, sodium dodecyl sulfate (SDS)-protein complexes are separated by sieving electrophoresis in a microfluidic device or chip. The chip is interfaced to a moving membrane so that proteins are captured in discrete zones as they migrate from the chip. Separations of SDS-protein complexes in the molecular weight range of 11-155 kDa were completed in 2 min with 4 × 10(4) theoretical plates at 460 V/cm. Migration time and peak area relative standard deviations were 3-6% and 0.2%, respectively. Detection limit for actin was 0.7 nM. Assays for actin, AMP-kinase, carbonic anhydrase, and lysozyme are shown to demonstrate versatility of the method. Total analysis time including immunoassay was 22-32 min for a single sample. Because processing membrane for immunoassay is the slow step of the assay, sequential injections from different reservoirs on the chip and capture in different tracks on the same membrane allow increased throughput. As a demonstration, 9 injections were collected on one membrane and analyzed in 43 min (~5 min/sample). Further improvements in throughput are possible with more reservoirs or parallel channels. PMID:23672369

Jin, Shi; Anderson, Gwendolyn J; Kennedy, Robert T



Non-denaturing gel electrophoresis system for the purification of membrane bound proteins  

SciTech Connect

A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using this method, we were able partially to purify an /sup 3/H-etorphine binding glycoprotein, from placental villus tissue, with an apparent molecular weight range of 60-70,000. The iodinated glycoprotein migrates in SDS PAGE with an apparent molecular weight of 63,000. This method may be useful for the isolation of membrane bound proteins, especially when an affinity ligand is not available.

Cavinato, A.G.; Macleod, R.M.; Ahmed, M.S.



Polyacrylamide gel electrophoresis analysis of ribosomal protein AT-L30 from an actinomycete genus, Streptosporangium.  


We analyzed the ribosomal AT-L30 proteins from 11 type strains of species belonging to the genus Streptosporangium. The electrophoretic mobilities of the AT-L30 preparations from these strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that they could be divided into three groups. The first group contained Streptosporangium viridogriseum, S. viridogriseum subsp. kofuense, and S. albidum, while the second group contained S. roseum, S. album, S. vulgare, S. nondiastaticum, S. fragile, S. violaceochromogenes, and S. amethystogenes. S. corrugatum was a member of the third group. These groups were completely consistent with Nonomura's previous classification, which was based on morphological criteria. The results of partial amino acid sequencing of AT-L30 preparations from several representative strains strongly supported the hypothesis that each of the three groups of the genus Streptosporangium merits separate generic status. PMID:1736963

Ochi, K; Miyadoh, S



Previsible silver staining of protein in electrophoresis gels with mass spectrometry compatibility.  


A convenient silver staining method for protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8 ng/band in approximately 1h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1h with a detection limit of 0.2 ng/band. PMID:18804088

Jin, Li-Tai; Li, Xiao-Kun; Cong, Wei-Tao; Hwang, Sun-Young; Choi, Jung-Kap



Muscle protein analysis. II. Two-dimensional electrophoresis of normal and diseased human skeletal muscle  

SciTech Connect

High-resolution two-dimensional electrophoresis was used to analyze the major proteins of normal and pathological human-muscle samples. The normal human-muscle pattern contains four myosin light chains: three that co-migrate with the myosin light chains from rabbit fast muscle (extensor digitorum longus), and one that co-migrates with the light chain 2 from rabbit slow muscle (soleus). Of seven Duchenne muscular dystrophy samples, four yielded patterns with decreased amounts of actin and myosin relative to normal muscle, while three samples gave patterns comparable to that for normal muscle. Six samples from patients with myotonic dystrophy also gave normal patterns. In nemaline rod myopathy, in contrast, the pattern was deficient in two of the fast-type myosin light chains.

Giometti, C.S. (Argonne National Lab., IL); Barany, M.; Danon, M.J.; Anderson, N.G.



A quantitative measure for protein conformational heterogeneity  

NASA Astrophysics Data System (ADS)

Conformational heterogeneity is a defining characteristic of proteins. Intrinsically disordered proteins (IDPs) and denatured state ensembles are extreme manifestations of this heterogeneity. Inferences regarding globule versus coil formation can be drawn from analysis of polymeric properties such as average size, shape, and density fluctuations. Here we introduce a new parameter to quantify the degree of conformational heterogeneity within an ensemble to complement polymeric descriptors. The design of this parameter is guided by the need to distinguish between systems that couple their unfolding-folding transitions with coil-to-globule transitions and those systems that undergo coil-to-globule transitions with no evidence of acquiring a homogeneous ensemble of conformations upon collapse. The approach is as follows: Each conformation in an ensemble is converted into a conformational vector where the elements are inter-residue distances. Similarity between pairs of conformations is quantified using the projection between the corresponding conformational vectors. An ensemble of conformations yields a distribution of pairwise projections, which is converted into a distribution of pairwise conformational dissimilarities. The first moment of this dissimilarity distribution is normalized against the first moment of the distribution obtained by comparing conformations from the ensemble of interest to conformations drawn from a Flory random coil model. The latter sets an upper bound on conformational heterogeneity thus ensuring that the proposed measure for intra-ensemble heterogeneity is properly calibrated and can be used to compare ensembles for different sequences and across different temperatures. The new measure of conformational heterogeneity will be useful in quantitative studies of coupled folding and binding of IDPs and in de novo sequence design efforts that are geared toward controlling the degree of heterogeneity in unbound forms of IDPs.

Lyle, Nicholas; Das, Rahul K.; Pappu, Rohit V.



Two-dimensional gel electrophoresis and immunoblotting of Campylobacter outer membrane proteins.  

PubMed Central

We characterized outer membrane proteins (OMPs) from selected Campylobacter jejuni, C. coli, and C. fetus strains by two-dimensional gel electrophoresis (2DGE), using isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and by immunoblotting with immune rabbit serum. The flagellar band with a molecular mass of 63 kilodaltons (kDa) demonstrated previously by one-dimensional SDS-PAGE was shown by 2DGE to consist of one or several charge trains, depending upon the species, strain, and type of preparation studied; each of the individual peptides was found to be antigenic by immunoblotting. In contrast, in all of the strains studied, the major OMP (43 to 44 kDa) of C.jejuni and C. coli consisted of a single isomeric form which was weakly immunogenic. Several minor proteins (29 to 31 kDa) were found to be strongly immunogenic by immunoblotting. C. fetus strains possessed two major OMPs of 45 to 47 kDa, each of which consisted of either a single isomer or a major isomer comprising at least 90% of the major OMP. Serum-resistant strains of C. fetus possessed an acid-labile 100-kDa glycoprotein (pI, 4.1) which was markedly diminished or absent in serum-sensitive strains. These 2DGE analyses provide information that is useful in taxonomic and epidemiologic studies and for the purification of surface antigens for the development of campylobacter vaccines and may also facilitate the identification of specific virulence factors. Images

Dunn, B E; Blaser, M J; Snyder, E L



[Cerebrospinal fluid proteins: II. Normal values of protein fractions obtained by electrophoresis (variations related to race, sex and age)].  


The total protein content and protein fractions, obtained by electrophoresis on cellulose acetate strips, of CSF collected from the cisterna magna of 213 patients (with no neurological diseases) were determined in order to verify variations related to race, sex and age, as well as to establish the proteinogram normal limits. No differences between caucasians and coloured persons were observed with respect to CSF total protein and protein fractions. Children (5 months to 11 years old) do not present differences related to sex or age, on their proteinogram. Children's CSF total protein, relative values of pre-albumin, alpha 1--and beta-globulins, absolute values of albumin, alfa 2--, beta--, tau --and gamma-globulins, differ from those found in adults. Differences between males and females in the normal CSF proteinogram were found in adults. As a consequence of these findings, the CSF proteinogram normal limits for children, males and females were separately established. In adults, statistically significant positive correlation between age and the 7 protein fractions when expressed in mg/100 ml were observed, as well as between age and total protein. Comparison of the results obtained in this research with those found in some publications was carried out and is briefly discussed. PMID:6870591

Vermes, L M



Glucose-induced changes of multiple mouse islet proteins analysed by two-dimensional gel electrophoresis and mass spectrometry  

Microsoft Academic Search

Aims\\/hypothesis  The aim of this study was to investigate molecular mechanisms of glucose-induced changes in islets of Langerhans by analysing global changes in protein patterns of islets exposed to elevated glucose concentrations.Methods  Islets were isolated from C57BL\\/6J mice and used either directly or after exposure to 11 mmol\\/l glucose for 24 h. Islet protein profiles were obtained by two-dimensional gel electrophoresis, and protein spots

M. Ahmed; P. Bergsten



Performing isoelectric focusing and simultaneous fractionation of proteins on a rotary valve followed by sodium dodecyl-polyacrylamide gel electrophoresis.  


In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl-polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE, the second-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed. PMID:23819755

Wang, Wei; Lu, Joann J; Gu, Congying; Zhou, Lei; Liu, Shaorong



Quantitative Estimation of Cellular Retinoic Acid-binding Protein Activity in Normal, Dysplastic, and Neoplastic Human Breast Tissue1  

Microsoft Academic Search

A technique for reproducible and quantitative determination of human cellular retinole acid-binding protein (CRABP) activity in breast tissue specimens is described. A multiphasic poly- acrylamide disc gel electrophoresis system (operative at pH 10.2) was adapted for this purpose. This technique allows, after incubation with tritiated retinoic acid (RA) overnight, the separation of the specific CRABP activity from the nonspecific serum-originated

Willy M. KÃ; Eva Geyer; Urs Eppenberger; Peter R. Huber


Two dimensional non-equilibrium pH gel electrophoresis mapping of cytosolic protein changes caused by dietary protein depletion in mouse liver  

Microsoft Academic Search

Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate the effects of dietary protein depletion on the protein composition of mouse liver cytosol. Analysing the cytosol from both normal and protein depleted liver, the position in gels of more than three hundred protein spots was determined. After 5 days of protein depletion, about 20% of the spots either

Pedro Mariano Sanllorenti; Jorge Rosenfeld; Virginia Paola Ronchi; Pascual Ferrara; Rubén Danilo Conde



Multiple Reaction Monitoring-based, Multiplexed, Absolute Quantitation of 45 Proteins in Human Plasma*  

PubMed Central

Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ([13C6]Arg or [13C6]Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.

Kuzyk, Michael A.; Smith, Derek; Yang, Juncong; Cross, Tyra J.; Jackson, Angela M.; Hardie, Darryl B.; Anderson, N. Leigh; Borchers, Christoph H.



Pinnacle: a fast, automatic and accurate method for detecting and quantifying protein spots in 2-dimensional gel electrophoresis data  

Microsoft Academic Search

Motivation: One of the key limitations for proteomic studies using 2- dimensional gel electrophoresis (2DE) is the lack of rapid, robust, and reproducible methods for detecting, matching, and quantifying protein spots. The most commonly used approaches involve first detecting spots and drawing spot boundaries on individual gels, then matching spots across gels, and finally quantifying each spot by calculating normalized

Jeffrey S. Morris; Brittan N. Clark; Howard B. Gutstein



The characterization of Nigerian varieties of pepper, Capsicum annuum and Capsicum frutescens by SDS polyacrylamide gel electrophoresis of seed proteins  

Microsoft Academic Search

The possibility of using electrophoresis to characterize varieties of pepper, Capsicum annuum and Capsicum frutescens cultivated in Nigeria was investigated. The SDS- polyacrylamide gel electropherogram of extracted total seed proteins of 10 breeding lines in each of the 6 varieties investigated, revealed a pattern in which 12 polypeptide bands with apparent molecular weight range of 22 to 98 kilodaltons could

P. G. C. Odeigah; B. Oboh; I. O. Aghalokpe



Proteins of the kidney microvillus membrane. Identification of subunits after sodium dodecylsullphate/polyacrylamide-gel electrophoresis.  

PubMed Central

The proteins of microvilli prepared from pig kidney were analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The typical pattern stained for protein revealed five major bands, four of which also stained for carbohydrate, and about 15 minor bands. For descriptive purposes the bands were designated numerically by their apparent molecular weights (X10(-3). Well-characterized proteins were identified with four of the five major bands. Dipeptidyl peptidase IV, a serine proteinase that may be specifically labelled with di-isopropyl [32P]phosphorofluoridate, was assigned to band 130. Aminopeptidase M was assigned to band 160, though when released from the membrane by a proteinase, this protein comprises three polypeptides each of lower apparent molecular weight than the native enzyme. Neutral endopeptidase can be assigned to band 95 and actin to band 42. The fifth major band (180) is an extrinsic glycoprotein that has not been identified with any microvillus enzyme activity. These four proteins contribute 21% of the microvillus-membrane protein. Kidney microvillus actin was characterized by a variety of properties and was similar to muscle actin. A computer analysis of the gel pattern indicates that it comprises 9.0% of the microvillus protein. Myosin is not present in the microvillus, but another protein associated with band 95, with properties that distinguish it from neutral endopeptidase, was tentatively identified as alpha-actinin. Alkaline phosphatase was identified as a monomeric polypeptide with an apparent molecular weight of 80000; it is a minor protein of the microvillus and is not discernible as a discrete band in the gel pattern. These and other results permit a model of the organization of the microvillus protein to be suggested. The computer program used has been deposited as Supplementary Publication SUP 50070 (12 pages) at the British Library Lending Division, Boston Spa. Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1976) 153,5. Images PLATE 1 PLATE 2

Booth, A G; Kenny, A J



Use of Two-Dimensional Gel Electrophoresis To Measure Changes in Synovial Fluid Proteins from Patients with Rheumatoid Arthritis Treated with Antibody to CD4  

Microsoft Academic Search

Synovial fluid proteins from microliter volumes of synovial fluid were resolved by two-dimensional poly- acrylamide gel electrophoresis and detected by silver staining to investigate the feasibility of using two- dimensional (2D) electrophoresis in the clinical research setting and provide global disease information of disease progression. Several hundred proteins could be resolved as spots, many of which displayed the characteristic pattern




Detection of C-reactive protein based on magnetic nanoparticles and capillary zone electrophoresis with laser-induced fluorescence detection.  


A simple and fast method based on magnetic nanoparticles (MNPs) and capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection was developed for the detection of C-reactive protein (CRP). To optimize the CZE conditions, several factors including buffer compositions, buffer ionic strength, buffer pH, applied voltage and capillary temperature have been examined. The optimal separation buffer selected was a 30mM sodium phosphate (PB) buffer, pH 8.0. The optimal CE applied voltage and temperature selected were 20kV and 35°C, respectively. The CZE profile of the MNP-1°Ab-CRP-2°Ab/FITC bioconjugates showed good reproducibility. One major peak was observed for the MNP bioconjugates. The quantitative analysis also showed good results. The coefficient of variation (CV%) for the major peak area was 8.7%, and the CV% for the major peak migration time was 2.5%. The linear range for CRP analysis was 10-150?g/mL, and the concentration limit of detection (LOD) was 9.2?g/mL. Non-specific interactions between bovine serum albumin (BSA) and the system can be prevented by including 10% (v/v) of human plasma in the binding buffers. The CE/LIF method might be helpful for analyzing high concentrations of CRP in a patient's plasma after an acute-phase inflammation. This new method demonstrated the possibility of using MNPs and CE/LIF for the detection of proteins, and provided information for the establishment of appropriate CE conditions. PMID:24075015

Lin, Yi-Jyun; Yang, Jian-Ying; Shu, Ting-Yu; Lin, Ting-Yu; Chen, Yen-Yi; Su, Mei-Yu; Li, Wen-Jie; Liu, Mine-Yine



Separation of acidic and basic proteins by capillary electrophoresis using gemini surfactants and gemini-capped nanoparticles as buffer additives  

Microsoft Academic Search

This paper demonstrated simultaneous separation of acidic and basic proteins using cationic gemini surfactants as buffer additives\\u000a in capillary electrophoresis. We showed that even at a low concentration (0.1 mmol·L?1) of alkanediyl-?,?-bis(dimethyloctadecylammonium bromide) (18-s-18), the wall adsorption of both acidic and basic proteins could be effectively suppressed under acidic conditions. Smaller\\u000a micelle size (e.g., s = 5–8) is more effective

Qian Liu; YanQing Li; YanMin Yang; ShouZhuo Yao



Method for the typing of Clostridium difficile based on polyacrylamide gel electrophoresis of (/sup 35/S)methionine-labeled proteins  

SciTech Connect

A typing method for Clostridium difficile based on the incorporation of (/sup 35/S)methionine into cellular proteins, their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their visualization by autoradiography is described. On analysis of the radiolabeled-protein profiles, nine distinct groups were observed (A to E and W to Z). The method, which is simple, reproducible, and readily expandable, has been applied in epidemiological studies to demonstrate cross-infection and hospital acquisition of C. difficile.

Tabaqchali, S.; O'Farrell, S.; Holland, D.; Silman, R.



Analysis of brain proteins in Alzheimer’s disease using high-resolution two-dimensional gel electrophoresis  

Microsoft Academic Search

Two-dimensional gel electrophoresis (2-DE), a method which can be used to analyze the expression of many proteins, is a promising and powerful approach which we have begun to use in the characterization of the complex pathologic processes in Alzheimer’s disease (AD). In the present study, a reliable 2-DE database of human brain proteins was created by improving the reproducibility of

T. Tsuji; S. Shimohama; S. Kamiya; T. Sazuka; O. Ohara



Green fluorescent protein as a quantitative tool  

Microsoft Academic Search

Manipulating the expression of a protein can provide a powerful tool for understanding its function, provided that the protein is expressed at physiologically-significant concentrations. We have developed a simple method to measure (1) the concentration of an overexpressed protein in single cells and (2) the covariation of particular physiological properties with a protein's expression. As an example of how this

Nicola J Hack; Brian Billups; Peter B Guthrie; John H Rogers; Elizabeth M Muir; Thomas N Parks; Stanley B Kater



Indirect UV detection as a non-selective detection method in the qualitative and quantitative analysis of heparin fragments by high-performance capillary electrophoresis.  


The application of capillary electrophoresis (CE) in combination with indirect UV detection for the qualitative and quantitative analysis of synthetic low-molecular-mass heparin fragments, at low pH, is described. It is demonstrated that, in contrast to direct UV detection, with indirect UV detection the signal obtained for various synthetic heparin pentasaccharides is nearly independent of their molecular structure. Moreover, the sensitivity of indirect UV detection is at least one order of magnitude higher than that of direct UV detection. CE-indirect UV detection for the qualitative and quantitative analysis of low-molecular-mass glycosaminoglycans was achieved by using 5 mM 5-sulphosalisylic acid, pH 3 or 5 mM 1,2,4-tricarboxybenzoic acid, pH 3.5 as electrophoresis buffer and chromophore. The technique is exemplified by the analysis of three pharmaceutical preparations of synthetic heparin pentasaccharides. The method employing indirect UV detection was validated with respect to repeatability, limit of detection, limit of quantitation, linearity, accuracy and ruggedness. In the indirect detection mode, the limit of detection for synthetic pentasaccharides is below 5 fmol, whereas the limit of quantitation is about 25 fmol. The method shows excellent repeatability and is linear in the femtomole-picomole range. Finally, it is demonstrated that the method is suitable for the analysis of various types of glycosaminoglycans. PMID:7921190

Damm, J B; Overklift, G T



Specific and general stress proteins in Bacillus subtilis - a two-dimensional protein electrophoresis study  

Microsoft Academic Search

A computer-aided analysis of high resolution two-dimensional polyacrylamide gels was used to investigate the changes in the protein synthesis profile in B. subtilis wild-type strains and sigB mutants in response to heat shock, salt and ethanol stress, and glucose or phosphate starvation. The data provided evidence that the induction of at least 42 general stress proteins absolutely required the alternative

Jtjrg Bernhardt; U. Volker; A. Volker; Haike Antelmann; Roland Schmid; Hiltraut Mach; Michael Hecker



Characterization of discontinuous buffer junctions using pH indicators in capillary electrophoresis for protein preconcentration.  


An effective sample preconcentration technique for proteins and peptides was recently developed using capillary electrophoresis (CE) with discontinuous buffers [C.A. Nesbitt, J.T.-M. Lo, K.K.-C. Yeung, J. Chromatogr. A 1073 (2005) 175]. Two buffers of different pH created a junction to trap the sample molecules at their isoelectric points and resulted in over 1000-fold preconcentration for myoglobin within 30 min. To study the formation of pH junctions in CE, a pH indicator, bromothymol blue, is used in this work to reveal the pH changes at the discontinuous buffer boundary. Bromothymol blue (BTB) exhibits a drastic change in its visible absorption spectrum (300-600 nm) going from the acidic to basic pH conditions, and is therefore ideal for visualizing the changes in pH at the junctions created by various buffer combinations. Preconcentration of myoglobin was performed in discontinuous buffers containing BTB. Major differences in the BTB absorption profiles were identified from buffer systems that differ significantly in preconcentration performance, which in turn, allowed for the identification of ideal buffers for sample preconcentration. Up to 2000-fold preconcentrations of myoglobin were achieved in the buffer systems studied in this work. In addition, the role of the electroosmotic flow (EOF) on the preconcentration performance was investigated. A low EOF was found to be desirable, as the pH junction could stay longer in the capillary for accumulation of proteins. The pH junction also displayed characteristics to resist bandbroadening. Potential laminar flow resulted from the mismatched residual EOFs under the two pH conditions within the discontinuous buffers appeared to have minimal effect on the preconcentration. In fact, external applied pressure can be used to control the migration of the pH junction without compromising the protein preconcentration. PMID:17022988

Jurcic, Kristina; Nesbitt, Chandra A; Yeung, Ken K-C



The Lowry Method for Protein Quantitation  

Microsoft Academic Search

\\u000a The most accurate method of determining protein concentration is probably acid hydrolysis followed by amino acid analysis.\\u000a Most other methods are sensitive to the amino acid composition of the protein, and absolute concentrations cannot be obtained.\\u000a The procedure of Lowry et al. (1) is no exception, but its sensitivity is moderately constant from protein to protein, and it has been

Jakob H. Waterborg; Harry R. Matthews


Determination of urinary protein fractions. A comparison with different electrophoretic methods and quantitatively determined protein concentrations.  


The second morning urine of 207 patients with various renal diseases was examined. After testing the urine with the Combur-9-RL((R)) dip strip, the concentrations of total protein, creatinine, albumin, alpha-1-microglobulin, immunoglobulin G and transferrin were determined. The urinary protein fractions were measured in parallel with the electrophoresis kits: Hydragel-Proteinuria (sebia GmbH, Fulda); Protur-HiSi (Beckman Coulter GmbH, Krefeld); SPE-II (Beckman Coulter) and the protein concentrations were calculated. The method comparison was based on the agreement of the ascertained types of proteinuria, and on comparison of the electrophoretically obtained albumin protein concentrations with the automated concentration results. It could be shown that urine electrophoresis is a reliable analytic method for the diagnosis of proteinurias. A good agreement and significant correlations were found between single protein determination and urinary electrophoresis. A diagnostic strategy that combines electrophoresis, single protein determination and immunofixation is useful for the diagnosis and differentiation of proteinuria. PMID:10841918

Umbreit, A; Wiedemann, G




Microsoft Academic Search

Radial immunodiffusion is used to determine specific proteins in friction blister fluid and cantharidin blister fluid of human volunteers. Four proteins (albumin, fibrinogen, immunoglobin IgG and IgM) were determined in blister fluid pooled 2, 4 and 21 hours after blistering. The same proteins were also determined in cantharidin blister fluid from the back and palm. The concentration of a specific

Peter Schmid



Endotoxin Quantitation by Measurement of Non-Precipitated Limulus Proteins.  

National Technical Information Service (NTIS)

Using the Limulus Amoebocyte Lysate test a number of procedures have been developed for the quantitation of endotoxin. This report introduces a new method based on the relationship between non-precipitated limulus proteins and endotoxin. Using this relati...

D. A. DuBose J. M. Brown



The Bradford Method for Protein Quantitation  

Microsoft Academic Search

\\u000a A rapid and accurate method for the estimation of protein concentration is essential in many fields of protein study. An assay\\u000a originally described by Bradford (1) has become the preferred method for quantifying protein in many laboratories. This technique is simpler, faster, and more\\u000a sensitive than the Lowry method. Moreover, when compared with the Lowry method, it is subject to

Nicholas J. Kruger


Quantitative microscopy: protein dynamics and membrane organisation.  


The mobility of membrane proteins is a critical determinant of their interaction capabilities and protein functions. The heterogeneity of cell membranes imparts different types of motion onto proteins; immobility, random Brownian motion, anomalous sub-diffusion, 'hop' or confined diffusion, or directed flow. Quantifying the motion of proteins therefore enables insights into the lateral organisation of cell membranes, particularly membrane microdomains with high viscosity such as lipid rafts. In this review, we examine the hypotheses and findings of three main techniques for analysing protein dynamics: fluorescence recovery after photobleaching, single particle tracking and fluorescence correlation spectroscopy. These techniques, and the physical models employed in data analysis, have become increasingly sophisticated and provide unprecedented details of the biophysical properties of protein dynamics and membrane domains in cell membranes. Yet despite these advances, there remain significant unknowns in the relationships between cholesterol-dependent lipid microdomains, protein-protein interactions, and the effect of the underlying cytoskeleton. New multi-dimensional microscopy approaches may afford greater temporal and spatial resolution resulting in more accurate quantification of protein and membrane dynamics in live cells. PMID:19416480

Owen, Dylan M; Williamson, David; Rentero, Carles; Gaus, Katharina



Aleutian disease serology, protein electrophoresis, and pathology of the European mink (Mustela lutreola) from Navarra, Spain.  


The European mink, Mustela lutreola, has suffered a dramatic decline in Europe during the 20th century and is one of the most endangered carnivores in the world. The subpopulation of European mink from Navarra, Spain, estimated to number approximately 420, represents approximately two thirds of the total number of mink in Spain. Aleutian Disease Virus (ADV) is a parvovirus with a high degree of variability that can infect a broad range of mustelid hosts. The pathogenesis of this virus in small carnivores is variable and can be influenced by both host factors (e.g., species, American mink genotype, and immune status) and viral strain. A cross-sectional study was conducted during the pre-reproductive period of February-March 2004 and 2005 and the postreproductive period of September-December 2004. Mink were intensively trapped along seven rivers that were representative of the European mink habitat in Navarra. Antibody counter immunoelectrophoresis against ADV was performed on 84 European mink blood samples. All the samples were negative. Protein electrophoresis was performed on 93 plasma samples. Nine of those samples (9.6%) had gamma globulin levels exceeding 20% of the total plasma protein. Complete necropsies were performed on 23 cadavers of European mink collected in the area between 2000 and 2005. Seventeen of the mink (74%) had traumatic and hemorrhagic lesions compatible with vehicular impact injuries. Although there were no histopathologic lesions associated with ADV, this study documents the first description of a naturally occurring canine distemper virus infection in a European mink. In addition, pulmonary adiaspiromycosis in three European mink from Spain was reported. PMID:18816991

Sánchez-Migallón Guzmán, David; Carvajal, Ana; García-Marín, Juan F; Ferreras, María C; Pérez, Valentín; Mitchell, Mark; Urra, Fermín; Ceña, Juan C



Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis.  


The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions. PMID:12833506

Manabe, Takashi; Yamaguchi, Nao; Mukai, Jun; Hamada, Osamu; Tani, Osamu



Capillary electrophoresis for purity estimation and in-process testing of recombinant GB virus-C proteins.  


Protein purity estimation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with scanning densitometry is a critical component in the manufacture of recombinant proteins for commercial diagnostic assays. However, the procedure is time consuming and often difficult to reproduce because commercial dyes that are used for visualizing proteins do not bind in a stoichiometric fashion for all proteins. The present report describes the use of a rapid and dye-independent SDS polymer-filled capillary gel electrophoresis (CE-SDS) method to estimate protein purity. The CE-SDS method was used for in-process and final purity testing of GB virus-C (GBV-C) fusion proteins produced in E. coli, and was directly compared with the conventional SDS-PAGE method using purified Coomassie blue dye to reduce protein staining anomalies. The CE-SDS method may serve as an alternative or replacement method to the lengthy and tedious SDS-PAGE method. This study also demonstrates that the observed molecular weight of the fusion protein, determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), provides higher accuracy than values estimated by either CE-SDS or SDS-PAGE methods. PMID:9384714

Kundu, S; Fenters, C; Lopez, M; Varma, A; Brackett, J; Kuemmerle, S; Hunt, J C


Initial proteome analysis of caffeine-induced proteins in Aspergillus tamarii using two-dimensional fluorescence difference gel electrophoresis.  


Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi. PMID:22391696

Gutiérrez-Sánchez, Gerardo; Atwood, James; Kolli, V S Kumar; Roussos, Sévastianos; Augur, Christopher



Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis.  


The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE. PMID:11245891

Williams, K M; Marshall, T



Simple, Time-Saving Dye Staining of Proteins for Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis Using Coomassie Blue  

Microsoft Academic Search

A fixation-free and fast protein-staining method for sodium dodecyl sulfate–polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye

Wei-Hua Dong; Tian-Yun Wang; Fang Wang; Jun-He Zhang



Quantitation of protein 3 content of circulating erythrocytes at the single-cell level  

SciTech Connect

The density and size of human erythrocytes has been roughly correlated with cell age, with the denser and smaller cells being older. Observations of this type have led to a hypothesis that the membranes of circulating erythrocytes are dynamic with respect to composition and that material is lost from the membrane during cell maturation and circulation. In this study, flow cytofluorimetry was used to investigate the distribution of the human erythrocyte anion transport protein (protein 3) in heterogeneous samples of circulating red cells. We verified that protein 3 can be specifically and quantitatively labeled in intact human erythrocytes with eosin-5-maleimide, a luminescent probe. Individual cells were accordingly analyzed for size by forward light scattering and for protein 3 content by quantitation of eosin fluorescence. Initial results indicated that the smallest erythrocytes had a protein 3 content equal to that of the largest circulating erythrocytes. This result was independently verified by light scatter-activated cell sorting; direct measurement of cell diameters by microscopy verified that the cell sizes of erythrocytes showing the 10% greatest and 10% smallest light-scattering signal were indeed distinct. Independent analysis of the size-sorted erythrocytes for protein 3 content was accomplished by gel electrophoresis of stroma from 150,000 large and small erythrocytes. Quantitative scanning densitometry of silver-stained gels of prepared stroma showed that protein 3 content of each set of fractionated cells was equal and did not vary as a function of cell size. Taken in combination with the reported correlation between increasing red blood cell age and decreasing cell size, these results indicate that any loss of membranous material during the cell aging process is not random.

Jennings, L.K.; Brown, L.K.; Dockter, M.E.



Phenols content and 2-D electrophoresis protein pattern: a promising tool to monitor Posidonia meadows health state  

PubMed Central

Background The endemic seagrass Posidonia oceanica (L.) Delile colonizes soft bottoms producing highly productive meadows that play a crucial role in coastal ecosystems dynamics. Human activities and natural events are responsible for a widespread meadows regression; to date the identification of "diagnostic" tools to monitor conservation status is a critical issue. In this study the feasibility of a novel tool to evaluate ecological impacts on Posidonia meadows has been tested. Quantification of a putative stress indicator, i.e. phenols content, has been coupled to 2-D electrophoretic protein analysis of rhizome samples. Results The overall expression pattern from Posidonia rhizome was determined using a preliminary proteomic approach, 437 protein spots were characterized by pI and molecular weight. We found that protein expression differs in samples belonging to sites with high or low phenols: 22 unique protein spots are peculiar of "low phenols" and 27 other spots characterize "high phenols" samples. Conclusion Posidonia showed phenols variations within the meadow, that probably reflect the heterogeneity of environmental pressures. In addition, comparison of the 2-D electrophoresis patterns allowed to highlight qualitative protein expression differences in response to these pressures. These differences may account for changes in metabolic/physiological pathways as adaptation to stress. A combined approach, based on phenols content determination and 2-D electrophoresis protein pattern, seems a promising tool to monitor Posidonia meadows health state.

Migliore, Luciana; Rotini, Alice; Randazzo, Davide; Albanese, Nadia N; Giallongo, Agata



The use of ASB-14 in combination with CHAPS is the best for solubilization of human brain proteins for two-dimensional gel electrophoresis  

Microsoft Academic Search

Protein extraction is the most important step to reveal a proteome by Two-Dimensional Gel Electrophoresis. Usually, the urea\\/thiourea based standard protein extraction buffer (SB) is combined with detergents with the aim of achieving better resolution and solubilization of different classes of proteins. In order to produce better gels and achieve the greatest spot resolution of Human Brain Proteins, comparisons using

Daniel Martins; Bruno Menezes de Oliveira; Santos Farias; Ricardo Shiniti; Oka Horiuchi; Cleyton Crepaldi Domingues; Eneida de Paula; Sergio Marangoni; Wagner Farid Gattaz; Emmanuel Dias-Neto



Simple, time-saving dye staining of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue.  


A fixation-free and fast protein-staining method for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye stain does not contain any amount of acid and methanol, such as phosphoric acid. Considering the speed, simplicity, and low cost, the dye stain may be of more practical value than other dye-based protein stains in routine proteomic research. PMID:21850222

Dong, Wei-Hua; Wang, Tian-Yun; Wang, Fang; Zhang, Jun-He



Proteome mapping by two-dimensional polyacrylamide gel electrophoresis in combination with mass spectrometric protein sequence analysis  

Microsoft Academic Search

\\u000a The high resolving power of two-dimensional polyacrylamide gel electrophoresis 2D-PAGE and its full analytical and preparative\\u000a potential have been described with special emphasis on reproducibility and standardization of protein spot patterns, enhanced\\u000a protein detection sensitivity, and computer analysis database development. New methodologies for peptide mass fingerprinting,\\u000a peptide, sequence, and fragmention tagging have been highlighted. Major challenges associated with 2D-PAGE\\/mass spectrometric

Ettore Appella; David Arnott; Kazuyasu Sakaguchi; Peter J. Wirth


A new multiphasic buffer system for benzyldimethyl-n-hexadecylammonium chloride polyacrylamide gel electrophoresis of proteins providing efficient stacking.  


Acidic PAGE systems using cationic detergents such as benzyldimethyl-n-hexadecylammonium chloride (16-BAC) or CTAB have proven useful for the detection of methoxy esters sensitive to alkaline pH, resolving basic proteins such as histones and membrane proteins. However, the interesting phosphate-based system suffered from poor stacking, resulting in broadened bands and long running times. Therefore, a new 16-BAC PAGE system based on the theory of moving boundary electrophoresis with properties comparable to the classical SDS-PAGE system was designed. As a result a new multiphasic analytical 16-BAC PAGE system providing efficient stacking and significantly shorter running times is presented here. It is based on acetic acid and methoxyacetic acid as common ion constituents. This PAGE system takes advantage of the additional counter stacking effect due to a cross boundary electrophoresis system resulting from the selected buffer constituents. Furthermore, the concentration of 16-BAC was optimized by determining its previously unknown CMC. Due to efficient focusing of the introduced tracking dye, methyl green, termination of electrophoresis can now be more easily followed as compared to the Schlieren line. PMID:16331586

Kramer, Michael L



Lithium Dodecyl Sulfate\\/Polyacrylamide Gel Electrophoresis of Thylakoid Membranes at 4 degrees C: Characterizations of Two Additional Chlorophyll A-Protein Complexes  

Microsoft Academic Search

Lithium dodecyl sulfate\\/polyacrylamide gel electrophoresis of Chlamydomonas reinhardtii thylakoid membranes at room temperature gave two chlorophyll-protein complexes, CP I and CP II, as had been reported previously. However, when the electrophoresis was performed at 4 degrees C, there was an increase in the amount of chlorophyll associated with CP I and CP II, and in addition, three other chlorophyll-protein complexes

Philippe Delepelaire; Nam-Hai Chua



Proteomic Analysis of Methylarginine-Containing Proteins in HeLa Cells by Two-Dimensional Gel Electrophoresis and Immunoblotting with a Methylarginine-Specific Antibody  

Microsoft Academic Search

Protein arginine methylation is found in many nucleic acid binding proteins affecting numerous cellular functions. In this\\u000a study we identified methylarginine-containing proteins in HeLa cell extracts by two-dimensional electrophoresis and immunoblotting\\u000a with a methylarginine-specific antibody. Protein spots with matched protein stain and blotting signals were analyzed by mass\\u000a spectrometry. The identities of 12 protein spots as 11 different proteins were

Chien-Jen Hung; Yu-Jen Lee; Da-Huang Chen; Chuan Li



Optimization and validation of a quantitative capillary electrophoresis sodium dodecyl sulfate method for quality control and stability monitoring of monoclonal antibodies.  


In previous work, a capillary electrophoresis sodium dodecyl sulfate (CE-SDS) method using precolumn labeling and laser-induced fluorescence (LIF) detection was developed at Genentech Inc. as part of the control system for the quality control release of a recombinant monoclonal antibody (rMAb) (Hunt, G.; Nashabeh, W. Anal. Chem. 1999, 71, 2390-2397.). In the current work, a generic and quantitative CE-SDS assay with LIF detection of rMAbs with improved accuracy and precision is described. The implementation of an alkylating step with iodoacetamide and optimization of the incubation temperature and time, in the presence of SDS, greatly decrease any thermally induced fragmentation of nonreduced labeled rMAb samples. In addition, a quantitative study of the effects of sample buffer pH on rMAb fragmentation is also presented. Furthermore, the performance of alternative CE-SDS polymer solutions and instrumentation for quantitative analysis of rMAbs is shown in this article. The validation of this method, under the guidelines of the International Committee on Harmonization (ICH), demonstrates that the assay quantitatively determines the consistency of rMAb manufacture as it relates to size heterogeneity and product purity. PMID:16970337

Salas-Solano, Oscar; Tomlinson, Brandon; Du, Sarah; Parker, Monica; Strahan, Alex; Ma, Stacey



Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics*  

PubMed Central

Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ?5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution.

Larance, Mark; Ahmad, Yasmeen; Kirkwood, Kathryn J.; Ly, Tony; Lamond, Angus I.



Capillary electrophoresis of peptides and proteins in acidic, isoelectric buffers: recent developments.  


The use of isoelectric buffers in capillary zone electrophoresis is here reviewed. Such buffers allow delivery of very high voltage gradients (up to 1000 V/cm in relatively large bore capillaries, e.g. 75-100 microm I.D.), permitting separations of the order of a few minutes and thus conserving (in fact favouring) very high resolution due to minimal, diffusion-driven, peak spreading. Isoelectric Asp (pI 2.77 at 50 mM concentration and 25 degrees C) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thus eliciting separation of unresolved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human beta globin chains. Also imino diacetic acid (pI 2.33 at 50 mM concentration) allows generation of high resolution peptide maps. Isoelectric Asp, in presence of 7 M urea and 0.5% hydroxyethyl cellulose (Mn = 27 000 Da) is also the preferred medium for fast separation and analysis of storage proteins in cereals, such as gliadins in soft and durum wheat and zeins in maize. A solution of 50 mM iminodiacetic acid (pI 2.23) containing 7 M urea and 0.5% hydroxyethylcellulose (apparent pH 3.2) is effectively used as background electrolyte for fast separation of heme-free, denatured globin (alpha and beta) chains. In the presence of neutral to neutral amino acid substitutions, it is additionally shown that the inclusion of 3% surfactant (Tween 20) in the sample and background electrolyte induces the separation of the wild-type and mutant chains, probably by a mechanism of hydrophobic interaction of the more hydrophobic mutant with the detergent micelle, via a mechanism similar to 'micellar electrokinetic chromatography'. PMID:10481947

Righetti, P G; Bossi, A; Olivieri, E; Gelfi, C



A Non-Denaturing Gel Electrophoresis System for the Purification of Membrane Bound Proteins  

Microsoft Academic Search

A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using

Anna G. Cavinato; Robert M. Macleod; Mahmoud S. Ahmed



Two-dimensional differential in-gel electrophoresis for identification of gastric cancer-specific protein markers.  


Gastric cancer is the second most common fatal malignancy in the world. Proteomics studies of clinical tumor samples have led to the identification of specific protein markers of gastric cancer detection and better understanding the carcinogenesis of gastric cancer. Gastric cancer tissue of epithelial origin and adjacent normal mucosa were examined in pair by fluorescence 2-D differential in-gel electrophoresis proteomics analysis utilizing 2-D PAGE protein separation. Intensity changes of 33 spots were detected with statistical significance. Twenty-two out of the 33 spots were identified by MALDI-TOF MS or MS/MS. Of the 9 up-regulated proteins, 7 were identified, including heat shock protein 60 (HSP60), mutant desmin, effector cell proteinase receptor 1 splice form 1b, hypothetical protein, unnamed protein product, and manganese superoxide dismutase (MnSOD), a protein similar to alpha-actin. Of the 20 down-regulated proteins, 16 were identified, including selenium binding protein 1, fibrinogen gamma, HSP27, tubulin alpha 6, zinc finger protein 160, prostaglandin F synthase, and eukaryotic translation elongation factor 1 alpha 1. Our results suggest that MnSOD may be a potential serum marker for molecular diagnosis of gastric carcinoma, and DIGE is a useful technique for screening differentially expressed proteins in cancer tissues. PMID:19424620

Wu, Cheng; Luo, Zhiwen; Chen, Xueyun; Wu, Chaoqun; Yao, Dingkang; Zhao, Peng; Liu, Lijie; Shi, Bin; Zhu, Liang



The APEX Quantitative Proteomics Tool: Generating protein quantitation estimates from LCMS\\/MS proteomics results  

Microsoft Academic Search

BACKGROUND: Mass spectrometry (MS) based label-free protein quantitation has mainly focused on analysis of ion peak heights and peptide spectral counts. Most analyses of tandem mass spectrometry (MS\\/MS) data begin with an enzymatic digestion of a complex protein mixture to generate smaller peptides that can be separated and identified by an MS\\/MS instrument. Peptide spectral counting techniques attempt to quantify

John C. Braisted; Srilatha Kuntumalla; Christine Vogel; Edward M. Marcotte; Alan R. Rodrigues; Rong Wang; Shih-ting Huang; Erik S. Ferlanti; Alexander I. Saeed; Robert D. Fleischmann; Scott N. Peterson; Rembert Pieper



Quantitative profiling and identification of differentially expressed plasma proteins in friedreich's ataxia.  


Friedreich's ataxia (FRDA) is an autosomal recessive ataxia, characterized by progressive gait ataxia, limb ataxia, dysarthria, and areflexia associated with diabetes and hypertrophic cardiomyopathy. The primary cause of FRDA is the presence of expanded DNA triplet (GAA) repeats in the first intron of the fxn gene on chromosome 9q13. The expanded DNA repeats in fxn inhibit expression of the protein frataxin, which leads to neuronal degeneration. The aim of the study was to identify differentially expressed plasma proteins in FRDA patients for their diagnostic/prognostic applications. Clinically suspected FRDA patients (n = 42) were assessed on the International Co-Operative Ataxia Rating Scale (ICARS), and genetic confirmation was performed by analyzing (GAA) repeats via PCR. Eighteen patients were confirmed to be homozygous for FRDA, with ICARS scores of 40 ± 8. Plasma proteomics of homozygous FRDA patients and age- and gender-matched healthy controls was done using two-dimensional difference in-gel electrophoresis and LC-MS/MS. Quantitative proteomic analysis (fold change ?1.5; P < 0.05) revealed 13 differentially expressed protein spots. These proteins were found to be associated with neuropathy (?1-antitrypsin), ataxia (apolipoprotein A-I), oxidative stress (albumin), altered lipid metabolism (apolipoprotein C-II, C-III), etc. Further investigations of these differentially expressed proteins can aid in identifying prognostic/diagnostic markers for FRDA. © 2013 Wiley Periodicals, Inc. PMID:23996585

Swarup, Vishnu; Srivastava, Achal K; Padma, Madakasira V; Rajeswari, Moganty R



Broad-spectrum Four-dimensional Orthogonal Electrophoresis: A Novel Comprehensively Feasible System for Protein Complexomics Investigation*  

PubMed Central

The major challenge of “protein complexomics” is to separate intact protein complexes or interactional proteins without dissociation or denaturation from complex biological samples and to characterize structural subunits of protein complexes. To address these issues, we developed a novel approach termed “broad-spectrum four-dimensional orthogonal electrophoresis (BS4-DE) system,” which is composed of a nondenaturing part I and denaturing part II. Here we developed a mild acidic-native-PAGE to constitute part I, together with native-thin-layer-IEF and basic-native-PAGE, widening the range of BS4-DE system application for extremely basic proteins with the range of pI from about 8 to 11 (there are obviously 1000 kinds of proteins in this interval), and also speculated on the mechanism of separating. We first proposed ammonium hydroxide-ultrasonic protein extractive strategy as a seamless connection between part I and part II, and also speculated on the extractive mechanism. More than 4000 protein complexes could be theoretically solved by this system. Using this approach, we focus on blood rich in protein complexes which make it challenging to sera/plasma proteome study. Our results indicated that the BS4-DE system could be applied to blood protein complexomics investigation, providing a comprehensively feasible approach for disease proteomics.

Wang, Xiaodong; Li, Fenjie; Song, Gaoguang; Guo, Shuai; Liu, Hui; Chen, Guoqiang; Li, Zhili



Purification of multisubunit membrane protein complexes: isolation of chloroplast FoF1-ATP synthase, CFo and CF1 by blue native electrophoresis.  


The proton-ATP synthase of thylakoid membranes from chloroplasts (CFoF1) is composed of two parts with different structural and functional properties: the membrane-integral, proton-conducting complex CFo and the hydrophilic part, CF1 which catalyze the formation of adenosine-5'-triphosphate (ATP). To date it is difficult to isolate functional CFoF1 from thylakoids in high purity and yield. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was therefore successfully employed to isolate CFoF1 in a one-step procedure from thylakoid membranes. Using a cathode buffer with low Coomassie Blue G-250 (CBG) concentration (0.002%), CFoF1 remains intact and can be obtained in high purity from solubilized, prepurified ATP synthase. Using BN-PAGE and a cathode buffer with 0.02% CBG, the ATP synthase bifurcates, and we were able to isolate both parts, CFo and CF1, separately. CFoF1, CFo, and CF1, respectively, were electroeluted nearly quantitatively electroeluted from the gel. BN-PAGE is a generally applicable method for the isolation and characterization of multisubunit membrane protein complexes in their native structure. However, the combination of neutral detergents and the negatively charged dye CBG seems to mimic properties of mild ionic detergents. This effect can lead to dissociation of labile subunits and subcomplexes, especially when delipidated membrane protein complexes are applied to BN-PAGE. By variation of the initial electrophoresis conditions, i.e., dye concentration in the cathode buffer, amount of lipid and detergent, BN-PAGE can be used for the isolation of either intact complexes or of subcomplexes. PMID:10364459

Neff, D; Dencher, N A



Protein Extraction Methods for Two-Dimensional Electrophoresis from Baphicacanthus cusia ( Nees) Bremek Leaves - A Medicinal Plant with High Contents of Interfering Compounds  

Microsoft Academic Search

Protein extraction is a critical step for two-dimensional electrophoresis (2-DE). Different plant samples require different and adaptive protein extraction protocols. The leaves of medicinal plant, Baphicacanthus cusia (Nees) Bremek are notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds (especially the secondary metabolites and pigments). This study was aimed to establish a routine procedure for

Xiao-liang XIANG; Shu-ju NING; Xia JIANG; Xiao-gui GONG; Ren-lei ZHU; Dao-zhi WEI



Quantitative imaging of lymphocyte membrane protein reorganization and signaling.  


Changes in membrane protein localization are critical to establishing cell polarity and regulating cell signaling. Fluorescence microscopy of labeled proteins allows visualization of these changes, but quantitative analysis is needed to study this aspect of cell signaling in full mechanistic detail. We have developed a novel approach for quantitative assessment of membrane protein redistribution based on four-dimensional video microscopy of fluorescently labeled proteins. Our analytic system provides robust automated methods for cell surface reconstruction, cell shape tracking, cell-surface distance measurement, and cluster formation analysis. These methods permit statistical analyses and testing of mechanistic hypotheses regarding cell signaling. We have used this approach to measure antigen-dependent clustering of signaling molecules in CD4+ T lymphocytes, obtaining clustering velocities consistent with single-particle tracking data. Our system captures quantitative differences in clustering between signaling proteins with distinct biological functions. Our methods can be generalized to a range of cell-signaling phenomena and enable novel applications not feasible with single-particle studies, such as analysis of subcellular protein localization in live organ culture. PMID:15501943

Kasson, Peter M; Huppa, Johannes B; Krogsgaard, Michelle; Davis, Mark M; Brunger, Axel T



Pressure-Assisted Capillary Electrophoresis Coupling with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometric Imaging for Quantitative Analysis of Complex Peptide Mixtures  

PubMed Central

Herein we report a pressure-assisted capillary electrophoresis-mass spectrometric imaging (PACE-MSI) platform for peptide analysis. This new platform has addressed the sample diffusion and peak splitting problems that appeared in our previous groove design, and it enables homogenous deposition of the CE trace for high-throughput MALDI imaging. In the coupling of CE to MSI, individual peaks (m/z) can be visualized as discrete colored image regions and extracted from the MS imaging data, thus eliminating issues with peak overlapping and reducing reliance on an ultra-high mass resolution mass spectrometer. Through a PACE separation, 46 tryptic peptides from bovine serum albumin and 150 putative neuropeptides from the pericardial organs of a model organism blue crab Callinectes sapidus were detected from the MALDI MS imaging traces, enabling four to six-fold increase of peptide coverage as compared with direct MALDI MS analysis. For the first time, quantitation with high accuracy was obtained using PACE-MSI for both digested tryptic peptides and endogenous neuropeptides from complex biological samples in combination with isotopic formaldehyde labeling. Although MSI is typically employed in tissue imaging, we show in this report that, it offers a unique tool for quantitative analysis of complex trace-level analytes with CE separation. These results demonstrate a great potential of the PACE-MSI platform for enhanced quantitative proteomics and neuropeptidomics.

Zhang, Zichuan; Ye, Hui; Wang, Junhua; Hui, Limei; Li, Lingjun



Cationic electrophoresis and eastern blotting.  


Denaturing, discontinuous electrophoresis in the presence of SDS has become a standard method for the protein scientist. However, there are situations where this method produces suboptimal results. In these cases, electrophoresis in the presence of positively charged detergents like cetyltrimethylammonium bromide may work considerably better. Methods for electrophoresis, staining, and blotting of such gels are presented. PMID:19378051

Buxbaum, Engelbert



Differentiation of hematuria by quantitative determination of urinary marker proteins  

Microsoft Academic Search

Summary Hematuria caused by prerenal, glomerular, postglomerular, and postrenal causes is usually differentiated by a number of noninvasive and invasive diagnostic procedures. In the present study we have applied a new analytical strategy based on observations that the various forms of hematuria can be classified by their typical protein pattern. When analyzed by quantitative turbidimetric assays, urines from postrenal hematurias

W. Hofmann; D. Schmidt; W. G. Guder; H. H. Edel



Protein Microchips in Quantitative Assays for Tumor Markers  

Microsoft Academic Search

Diagnosing malignant tumors is a major problem in oncology. The use of microchips in clinical testing makes it possible to detect several tumor markers in parallel without consuming large volumes of samples and expensive reagents. The goal of this study was to develop a quantitative immunoassay for some markers of commonly occurring tumors using three-dimensional hydrogel-based protein microchips designed at

E. I. Dementieva; A. Yu. Rubina; E. L. Darii; V. I. Dyukova; A. S. Zasedatelev; T. V. Osipova; T. P. Ryabykh; A. Yu. Baryshnikov; A. D. Mirzabekov



Assessing association between protein truncating variants and quantitative traits  

PubMed Central

Motivation: In sequencing studies of common diseases and quantitative traits, power to test rare and low frequency variants individually is weak. To improve power, a common approach is to combine statistical evidence from several genetic variants in a region. Major challenges are how to do the combining and which statistical framework to use. General approaches for testing association between rare variants and quantitative traits include aggregating genotypes and trait values, referred to as ‘collapsing’, or using a score-based variance component test. However, little attention has been paid to alternative models tailored for protein truncating variants. Recent studies have highlighted the important role that protein truncating variants, commonly referred to as ‘loss of function’ variants, may have on disease susceptibility and quantitative levels of biomarkers. We propose a Bayesian modelling framework for the analysis of protein truncating variants and quantitative traits. Results: Our simulation results show that our models have an advantage over the commonly used methods. We apply our models to sequence and exome-array data and discover strong evidence of association between low plasma triglyceride levels and protein truncating variants at APOC3 (Apolipoprotein C3). Availability: Software is available from Contact:

Rivas, Manuel A.; Pirinen, Matti; Neville, Matthew J.; Gaulton, Kyle J.; Moutsianas, Loukas; Lindgren, Cecilia M.; Karpe, Fredrik; McCarthy, Mark I.; Donnelly, Peter



Heterogeneity of a labeled tumor surface protein from a murine lung carcinoma demonstrated by two-dimensional electrophoresis  

SciTech Connect

Heterogeneity of a tumor surface protein (designated TSP-180) has been demonstrated by two-dimensional electrophoresis. Line 1 carcinoma cells derived from a spontaneous alveolar carcinoma of BALB/c mice were labeled externally with /sup 125/I by use of lactoperoxidase or metabolically with (/sup 3/H)-leucine before cell proteins were solubilized with Triton X-100 detergent. Immunoprecipitates prepared with heterologous antisera allowed comparison of two-dimensional patterns of line 1 surface proteins labeled with /sup 125/I or /sup 3/H. The isoelectric point of /sup 125/I-labeled TSP-180 was heterogeneous and varied between 6.1 and 6.3. Treatment with neuraminidase shifted the pI values to between 5.9 and 6.1 and reduced, but did not eliminate, the banding heterogeneity. These data show that charge heterogeneity due to sialization, as well as other factors, exists in TSP-180.

Eisinger, R.W. (Univ. of Tennessee, Oak Ridge); Kennel, S.J.



Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry  

Microsoft Academic Search

Background  Wheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the\\u000a abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers\\u000a with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use\\u000a MS\\/MS to distinguish the

Frances M Dupont; William H Vensel; Charlene K Tanaka; William J Hurkman; Susan B Altenbach



Gold nanoparticles amplified ultrasensitive quantification of human urinary protein by capillary electrophoresis with on-line inductively coupled plasma mass spectroscopic detection.  


Quantitative analysis of proteins play pivotal roles in basic discovery research and clinical applications, and the analytical challenge is to provide sufficient sensitivity to determine the proteins at endogenous levels. Here, we report a strategy for ultrasensitive quantification of human urinary protein by capillary electrophoresis with on-line inductively coupled plasma mass spectroscopic detection (CE-ICPMS) in conjunction with gold nanoparticles (AuNPs) amplification. The albumin in the sample solution was incubated with excess AuNPs to form the AuNP-albumin adduct. The excess AuNPs and the AuNP-albumin adduct were then effectively separated by CE for on-line ICPMS detection. As a result of AuNPs-tagging, more than 2000 gold atoms on average were attached to each albumin molecule to successfully achieve a significant amplification of ICPMS signal with extremely low limit of detection (0.5 pM for 280 nL of sample injection, corresponding to 0.1 amol) and a wide linear response over 4 orders of magnitude. The relative standard deviations of the migration time, peak area, and peak height for seven replicate injections of a mixture of 0.4 pM AuNPs and 9.0 pM albumin ranged from 1.8% to 4.4%. The developed method was successfully applied for detecting albumin in human urine samples with quantitative recoveries in the range of 93.0-99.7%. The methodology demonstrated here has potential for simultaneous determination of low-abundance multiple biomarkers of interest via multiple nanomaterials tags because of high-resolution CE separation and ultrasensitive ICPMS detection. PMID:20450228

Liu, Jing-Min; Li, Yan; Jiang, Yan; Yan, Xiu-Ping



Electrophoresis experiments for space  

NASA Astrophysics Data System (ADS)

It has long been hoped that space could alleviate the problems of large-scale, high-capacity electrophoresis. Support media and reduced chamber dimensions of capillary electrophoresis have established the physical boundaries for Earth-based systems. Ideally, electrophoresis conducted in a virtual weightless environment in an unrestricted ``free'' fluid should have great potential. The electrophoresis and isoelectric focusing experiments done in the reduced gravity over the past twenty-five years have demonstrated the absence of thermal convection and sedimentation as well as the presence of electrohydrodynamics that requires careful control. One commercial venture produced gram amounts of an electrophoretically purified protein during seven Space Shuttle flights but the market disappeared in the six years between experiment conception and performance on the Space Shuttle. Our accumulated experience in microgravity plus theoretical models predict improvements that should be possible with electrophoresis if past problems are considered and both invention of new technologies and innovation of procedures on the Space Station are encouraged. .

Snyder, Robert S.; Rhodes, Percy H.



Quantitation of carcinogen bound protein adducts by fluorescence measurements  

NASA Astrophysics Data System (ADS)

A highly significant correlation of aflatoxin B 1 serum albumin adduct level with daily aflatoxin B 1 intake was observed in a molecular epidemiological study of aflatoxin carcinogenesis which used conventional fluorescence spectroscopy methods for adduct quantitation. Synchronous fluorescence spectroscopy and laser induced fluorescence techniques have been employed to quantitate antibenzo[ a]pyrene diol epoxide derived globin peptide adducts. Fast and efficient methods to isolate the peptide adducts as well as eliminate protein fluorescence background are described. A detection limit of several femtomoles has been achieved. Experimental and technical considerations of low temperature synchronous fluorescence spectroscopy and fluorescence line narrowing to improve the detection sensitivities are also presented.

Gan, Liang-Shang; Otteson, Michael S.; Doxtader, Mark M.; Skipper, Paul L.; Dasari, Ramachandra R.; Tannenbaum, Steven R.



A rapid standardized quantitative microfluidic system approach for evaluating human tear proteins  

PubMed Central

Purpose To explore the potential of a chip-based miniaturized capillary gel electrophoresis device in a quantitative evaluation of the human tear protein profile and to validate the method. Methods A total of 5 ?l of tears were collected from 25 patients diagnosed as having mild to moderate dry eye according to Dry Eye Workshop guidelines and from 20 matched normal volunteers. Protein analysis was performed with the 2100 Bioanalyzer; different protein kit assays were evaluated (Protein 80 kit, Protein 230 kit, High Sensitivity Protein 250 kit) for sizing and quantifying protein samples from 5 to 80 kDa, 14 to 230 kDa, and 5 to 250 kDa, respectively. A standard protein ladder was loaded on each chip to allow an estimation of the appropriate molecular weight of the separated proteins; a sample buffer containing a lower and an upper marker was used to check the correct alignment of each lane. Virtual bands generated by the Bioanalyzer were identified and validated as follows: tear samples were run in parallel and proteins separated by one-dimensional and two-dimensional sodium dodecyl sulfate–PAGE and characterized by immunoblotting, enzymatic digestion, and analysis with liquid chromatography-mass spectrometry followed by a search of the SProt human protein database. Results Analyses were successfully performed by using as small as a 2 ?l tear sample. The Protein 230 kit was selected as the best chip kit, able to differentiate all the proteins of interest. The measurement noise parameters were low, and reproducibility and repeatability exhibited high accuracy (0.998 and 0.995, respectively) and precision (0.974 and 0.977, respectively). The coefficient of variability was slightly higher than that declared by the manufacturer (6.2% versus 5.0%). Total protein content and the following proteins were recognized in all samples: lipophilin A lysozyme C, tear lipocalin-1, zinc-alpha-2-glycoprotein, serotransferrin, lactotransferrin, and exudated serum albumin. Conclusions Our data demonstrate that this chip-based tear protein analysis is a reliable method of instrumental diagnosis in daily clinical activity and may provide supporting evaluation parameters for diagnosing and managing tear-based disorders.

Bavelloni, Alberto; Blalock, William; Fresina, Michela; Campos, Emilio C.



Milk Serum Proteins. I. A Quantitative Biuret Test for Milk Serum Proteins1  

Microsoft Academic Search

The biuret reaction has been used for many years as a qualitative test for the presence of proteins in solution. It depends on the formation of a violet copper- protein complex in alkaline CuSQ solution. This reaction first was adapted as a quantitative test for protein by Autenrieth (1, 2), who determined the albumin and globulin in urine, ascitic fluid

B. C. Johnson; A. M. Swanson



Visual integration of quantitative proteomic data, pathways and protein interactions  

PubMed Central

We introduce several novel visualization and interaction paradigms for visual analysis of published protein-protein interaction networks, canonical signaling pathway models, and quantitative proteomic data. We evaluate them anecdotally with domain scientists to demonstrate their ability to accelerate the proteomic analysis process. Our results suggest that structuring protein interaction networks around canonical signaling pathway models, exploring pathways globally and locally at the same time, and driving the analysis primarily by the experimental data, all accelerate the understanding of protein pathways. Concrete proteomic discoveries within T-cells, mast cells, and the insulin signaling pathway validate the findings. The aim of the paper is to introduce novel protein network visualization paradigms and anecdotally assess the opportunity of incorporating them into established proteomic applications. We also make available a prototype implementation of our methods, to be used and evaluated by the proteomic community.

Jianu, Radu; Yu, Kebing; Cao, Lulu; Nguyen, Vinh; Salomon, Arthur R.; Laidlaw, David H.



Protein quantitation using various modes of high performance liquid chromatography.  


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 (<15?g/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

Grotefend, Sandra; Kaminski, Lukas; Wroblewitz, Stefanie; Deeb, Sami El; Kühn, Nancy; Reichl, Stephan; Limberger, Markus; Watt, Steven; Wätzig, Hermann



Capillary electrophoresis-based profiling and quantitation of total salicylic acid and related phenolics for analysis of early signaling in Arabidopsis disease resistance.  


A capillary electrophoresis-based method for quantitation of total salicylic acid levels in Arabidopsis leaves was developed. Direct comparison to previous high-performance liquid chromatography (HPLC)-based measurements showed similar levels of salicylic acid. Simultaneous quantitation of trans-cinnamic acid, benzoic acid, sinapic acid, and an internal recovery standard was achieved. A rapid, streamlined protocol with requirements for plant tissue reduced relative to those of HPLC-based protocols is presented. Complicated, multiparameter experiments were thus possible despite the labor-intensive nature of inoculating plants with bacterial pathogens. As an example of this sort of experiment, detailed time course studies of total salicylic acid accumulation by wild-type Arabidopsis and two lines with mutations affecting salicylic acid accumulation in response to either of two avirulent bacterial strains were performed. Accumulation in the first 12h was biphasic. The first phase was partially SID2 and NDR1 dependent with both bacterial strains. The second phase was largely independent of both genes with bacteria carrying avrB, but dependent upon both genes with bacteria carrying avrRpt2. Virulent bacteria did not elicit salicylic acid accumulation at these time points. Application of this method to various Arabidopsis pathosystems and the wealth of available disease resistance signaling mutants will refine knowledge of disease resistance and associated signal transduction. PMID:12927828

Shapiro, Allan D; Gutsche, Annie Tang



An improved plant leaf protein extraction method for high resolution two-dimensional polyacrylamide gel electrophoresis and comparative proteomics.  


We report here a simple and universally applicable protocol for extracting high quality proteins from plant leaf tissues. The protocol provides improved resolution and reproducibility of two-dimensional polyacrylamide gel electrophoresis (2-DE) and reduces the time required to analyze samples. Partitioning rubisco by polyethylene glycol (PEG) fractionation provides clearer detection of low-abundance proteins. Co-extraction of interfering substances increases the sample conductivity, which results in poor electrophoretic separation. Re-extraction of PEG-fractionated samples with phenol effectively eliminated interfering substances, which results in optimal conductivity during separation in the first dimension of the isoelectric focusing. Smooth focusing reduces analysis time and provides superior resolution in 2-DE gels. Incubating the samples at -80° C instead of -20° C reduced protein precipitation time to 2-3 h. Removal of nonprotein contaminants and the use of sonication increased protein solubility without additional reagents. These changes enabled loading and separation of maximum amounts of proteins, which permitted improved protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). An immunological approach revealed that little or no ribulose-1, 5-bisphosphte bisphosphate carboxylase oxygenase was present in the PEG supernatant. In addition, low-abundance proteins, such as myelocytomatosis transcription factor (MYC) and alpha subunit of heterotrimeric guanine nucleotide-binding protein complex (G?), were detected only in the modified PEG supernatant and not in the total protein. These results suggest that our protocol produced high quality proteins and made many low-abundant proteins available for proteomic analysis. The successful application of this protocol for analyzing the leaf proteomes of soybean, Miscanthus sinensis, barley, Chinese cabbage, peanut and tea (Camellia sinensis) suggests that it could be used for comparative proteomic analysis of a wide range of plant leaves. PMID:23072551

Alam, I; Sharmin, Sa; Kim, K-H; Kim, Y-G; Lee, Jj; Lee, B-H



Optimization of protein separation by continuous-flow electrophoresis: influence of the operating conditions and the chamber thickness.  


The separation of protein by continuous-flow electrophoresis is disturbed by a number of effects which cause spreading of the protein streams with a loss of resolution. Two numerical models have been used to describe the coupling between the various spreading phenomena. A simple model takes into account molecular diffusion, electroosmosis and residence time gradients; the main model differs from the simple one by taking account of electrohydrodynamics. The influence of the separation chamber thickness, the radius of the sample filament, the field strength, and the residence time is explored. The simulations show that each dispersive effect has its own range of predominance, so that an optimum is found for the thickness of the separation chamber. PMID:10546810

Afonso, J L; Clifton, M J



Multilayer polymer microchip capillary array electrophoresis devices with integrated on-chip labeling for high-throughput protein analysis  

PubMed Central

It is desirable to have inexpensive, high-throughput systems that integrate multiple sample analysis processes and procedures, for applications in biology, chemical analysis, drug discovery, and disease screening. In this paper, we demonstrate multilayer polymer microfluidic devices with integrated on-chip labeling and parallel electrophoretic separation of up to 8 samples. Microchannels were distributed in two different layers and connected through interlayer through-holes in the middle layer. A single set of electrophoresis reservoirs and one fluorescent label reservoir address parallel analysis units for up to 8 samples. Individual proteins and a mixture of cancer biomarkers have been successfully labeled on-chip and separated in parallel with this system. A detection limit of 600 ng/mL was obtained for heat shock protein 90. Our integrated on-chip labeling microdevices show great potential for low-cost, simplified, rapid and high-throughput analysis.

Yu, Ming; Wang, Qingsong; Patterson, James E.; Woolley, Adam T.



Fast and selective determination of total protein in milk powder via titration of moving reaction boundary electrophoresis.  


In this paper, moving reaction boundary titration (MRBT) was developed for rapid and accurate quantification of total protein in infant milk powder, from the concept of moving reaction boundary (MRB) electrophoresis. In the method, the MRB was formed by the hydroxide ions and the acidic residues of milk proteins immobilized via cross-linked polyacrylamide gel (PAG), an acid-base indicator was used to denote the boundary motion. As a proof of concept, we chose five brands of infant milk powders to study the feasibility of MRBT method. The calibration curve of MRB velocity versus logarithmic total protein content of infant milk powder sample was established based on the visual signal of MRB motion as a function of logarithmic milk protein content. Weak influence of nonprotein nitrogen (NPN) reagents (e.g., melamine and urea) on MRBT method was observed, due to the fact that MRB was formed with hydroxide ions and the acidic residues of captured milk proteins, rather than the alkaline residues or the NPN reagents added. The total protein contents in infant milk powder samples detected via the MRBT method were in good agreement with those achieved by the classic Kjeldahl method. In addition, the developed method had much faster measuring speed compared with the Kjeldahl method. PMID:23483553

Guo, Cheng-ye; Wang, Hou-yu; Liu, Xiao-ping; Fan, Liu-yin; Zhang, Lei; Cao, Cheng-xi



Quartz crystal microbalances for quantitative biosensing and characterizing protein multilayers  

Microsoft Academic Search

The use of quartz crystal microbalances (QCMs) for quantitative biosensing and characterization of protein multilayers is demonstrated in three case studies. Monolayers of QCM-based affinity biosensors were investigated first. Layers of a thiol-containing synthetic peptide constituting an epitope of the foot-and-mouse-disease virus were formed on gold electrodes via self-assembly. The binding of specific antibodies to epitope-modified gold electrodes was detected

Jan Rickert; Andreas Brecht; Wolfgang Göpel



The Multi-Q web server for multiplexed protein quantitation  

Microsoft Academic Search

Multi-Q Web Server provides an automated data analysis tool for multiplexed protein quantitation based on the iTRAQ labeling method. Multi-Q is designed as a generic platform that can accommodate various input data formats from search engines and mass spectrometer manufacturers. In comparison with its previous stand-alone version, this new web server version provides many enhanced features and flexible options for

Chuan-yih Yu; Yin-hao Tsui; Yi-hwa Yian; Ting-yi Sung; Wen-lian Hsu



Increase in local protein concentration by field-inversion gel electrophoresis  

Microsoft Academic Search

BACKGROUND: Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing

Henghang Tsai; Teck Yew Low; Steve Freeby; Aran Paulus; Kalpana Ramnarayanan; Chung-pui Paul Cheng; Hon-chiu Eastwood Leung



Disc polyacrylamide gel electrophoresis of pollen proteins in the oil palm (Elaeis)  

Microsoft Academic Search

A preliminary screening of proteins and isozymes in the oil palm was investigated with a view to using the data in discriminating oil palm fruit forms. Protein and enzyme staining was carried out using pollen tissue. Repeatable bands of proteins which showed reproducable variation in banding patterns were obtained. Low percentage similarities between the groups compared for protein banding patterns

C. D. Ataga; C. A. Fatokun



Quantitating protein synthesis, degradation, and endogenous antigen processing.  


Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate. PMID:12648452

Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W



Protein Extraction for Two-Dimensional Gel Electrophoresis of Proteomic Profiling in Turfgrass  

Microsoft Academic Search

Protein extraction for two-dimensional gel elec- trophoresis (2-DE) from plant samples is chal- lenging due to low protein content and high level of contaminants. Proteomic research in turfgrass is limited by the lack of effi cient protein extrac- tion methods. To establish an effective protocol of protein extraction suitable for 2-DE analysis in turfgrasses, four protein extraction meth- ods (chloroform\\/acetone,

Chenping Xu; Yan Xu; Bingru Huang



Interactions of tumour-targeting nanoparticles with proteins: potential of using capillary electrophoresis as a direct probe.  


Metal-based nanoscale particles possess unique optoelectronic or magnetic properties that make them highly promising as imaging agents in cancer therapy research. The fate of nanoparticles in vivo and particularly, the delivery to tumours are closely related to their interactions with plasma proteins. Furthermore, proteins can be used to modify the nanoparticle surface in order to facilitate active targeting to tumours. Therefore, there is an ongoing need for new and more capable analytical methodologies to characterize the protein-nanoparticle binding. Due to the small-sample volume requirement, high degree of resolution and, most importantly, mild, species-friendly separation conditions, capillary electrophoresis (CE) is gaining increasing popularity in the analysis of protein-nanoparticle interaction. This perspective article highlights the potential of CE in studying reactions associated with protein-mediated transformations of nanoparticles, with the focus on quantum dots, gold and iron oxide nanoparticles. Different ways by which CE can be applied to such monitoring are summarized and critically assessed using a representative coverage of recent publications. PMID:23011516

Aleksenko, Svetlana S; Shmykov, Alexei Y; Oszwa?dowski, S?awomir; Timerbaev, Andrei R



Capillary electrophoresis-mass spectrometry of proteins at medium pH using bilayer-coated capillaries.  


The feasibility of using noncovalently bilayer-coated capillaries for capillary electrophoresis-mass spectrometry (CE-MS) of acidic proteins was investigated using background electrolytes (BGEs) of medium pH. The capillary was coated by successively rinsing the capillary with solutions of the oppositely charged polymers polybrene (PB) and poly(vinyl sulfonic acid) (PVS). Volatile BGEs containing ammonium formate and/or N-methyl morpholine were tested at pH 7.5 and 8.5. Overall, these BGEs provided relatively fast protein separations (analysis times of ca. 12 min) and showed high efficiencies (70,000-300,000 plates) when the ionic strength was sufficiently high. Migration-time reproducibilities were very favorable with RSDs of less than 1.0%. Infusion experiments showed satisfactory MS responses for studied proteins dissolved in ammonium formate (pH 8.5), however, high concentrations of N-methyl morpholine appeared to seriously suppress the MS protein signals. Evaluation of the CE-MS system was performed by analyzing a mixture of intact proteins yielding efficient separations and good-quality mass spectra. CE-MS analysis of a reconstituted formulation of the biopharmaceutical recombinant human growth hormone (rhGH) which was stored for a prolonged time, revealed one degradation product which was provisionally identified as desamido rhGH. Based on the MS responses the amount of degradation was estimated to be ca. 25%. PMID:17180183

Catai, Jonatan R; Toraño, Javier Sastre; de Jong, Gerhardus J; Somsen, Govert W



Fluorescent derivatization method of proteins for characterization by capillary electrophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection.  


A fast and improved sample preparation scheme was developed for protein analysis using capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) with laser-induced fluorescence detection. This CE-SDS method was developed as a purity assay for recombinant monoclonal antibodies (rMAbs). In this assay, rMAbs are derivatized with the fluorogenic reagent 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ) in the presence of a nucleophile (CN-), which fluoresces only upon covalent binding to the protein. Purification after labeling is therefore not necessary to remove unreacted reagents. Proteins are incubated at 75 degrees C for 5 min to facilitate denaturation and labeling. For nonreduced preparation, rMAbs are labeled at pH 6.5 with a dye-to-protein (D/P) molar ratio of 50:1, which forms conjugates having 6 +/- 4 FQ labels. For reduced preparation, rMAbs are labeled at pH 9.3 with a D/P molar ratio of 10:1, which generates light chain conjugates incorporated with 3 +/- 2 FQ labels. Labeling artifacts such as fragmentation or aggregation are absent with use of alkylation reagents. This efficient labeling scheme generates detection limits for FQ-labeled rMAbs as low as 10 ng/mL. In comparison to other labeling strategies, labeling proteins with FQ has the advantage of speed, ease of use, and robust quantification. PMID:17591753

Michels, David A; Brady, Lowell J; Guo, Amy; Balland, Alain



Electrolytic Reduction: Modification of Proteins Occurring in Isoelectric Focusing Electrophoresis and in Electrolytic Reactions in the Presence of High Salts  

PubMed Central

Artifacts in two-dimensional electrophoresis (2-DE) caused by the presence of salts in isoelectric focusing (IEF) have been previously described as a result of increasing conductivity and inducing electroosmosis. However, electrolysis induced by the presence of salts should not be disregarded. In this study, electrolytic reduction?oxidation reaction (redox) was found to be enhanced in the presence of salts in IEF. The consequence of the electrolytic redox leads to acidification of the low-pH region and alkalization of the high-pH region within the immobilized pH gradient (IPG) strip. As a result, a breakdown of immobilized pH buffer near the high pH region of IPG strips along with reduction of basic proteins resulted in uncharacterized artifacts in 2-DE. Electrolytic reduction in the presence of alkali and alkaline metal ions was demonstrated to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protein disulfide bonds, and protein carboxylic acids. Importantly, semipreparative electrolytic reduction of proteins can be carried out in the presence of sodium ions in a homemade electrolytic apparatus. These findings give additional explanations to the observed artifacts in 2-DE and reveal the unknown effects of salts in IEF. Moreover, we have provided a method with the potential to convert proteins or peptides to corresponding modified products containing aldehyde groups that can be used for conjugation with amine-containing compounds.



Analysis of aldosterone-induced differential receptor-independent protein patterns using 2D-electrophoresis and mass spectrometry.  


In the human body the mineralocorticoid aldosterone is responsible for maintaining water and electrolyte homeostasis and therefore controlling blood pressure. In addition, aldosterone has recently been associated with severe heart failure. Besides receptor-dependent action, the damaging effects of aldosterone may also be partly mediated through non-genomic mechanisms. The present study focuses on the mineralocorticoid receptor-independent action of aldosterone at the protein level. We chose the fission yeast Schizosaccharomyces pombe as a model organism, since this yeast does not contain nuclear steroid receptors, but many genes and regulatory mechanisms that are close to those of mammals. Using 2D-electrophoresis we identified for the first time protein spots affected by aldosterone in a nuclear receptor-free system. Mass spectrometry analysis using MALDI-TOF MS and nanoLC-MS/MS approaches allowed the unambiguous identification of 11 proteins that showed increased or decreased levels, which may represent newly identified players and pathways of aldosterone-induced action. Two proteins with a connection to osmotic regulation (NAD-dependent malic enzyme and glycerol-3-phosphate-dehydrogenase), as well as two proteins involved in the overall organization of the cytoskeleton, vip1 and glyceraldehyde-3-phosphate dehydrogenase, which was also found to be specifically affected by aldosterone in human HCT116 cells, are discussed. PMID:16913842

Böhmer, Susanne; Carapito, Christine; Wilzewski, Britta; Leize, Emmanuelle; Van Dorsselaer, Alain; Bernhardt, Rita



Molecular phylogeny of the hominoid primates as indicated by two-dimensional protein electrophoresis  

SciTech Connect

A molecular phylogeny for the hominoid primates was constructed by using genetic distances from a survey of 383 radiolabeled fibroblast polypeptides resolved by two-dimensional electrophoresis (2DE). An internally consistent matrix of Nei genetic distances was generated on the basis of variants in electrophoretic position. The derived phylogenetic tree indicated a branching sequence, from oldest to most recent, of cercopithecoids (Macaca fascicularis), gibbon-siamang, orangutan, gorilla, and human-chimpanzee. A cladistic analysis of 240 electrophoretic characters that varied between ape species produced an identical tree. Genetic distance measures obtained by 2DE are largely consistent with those generated by other molecular procedures. In addition, the 2DE data set appears to resolve the human-chimpanzee-gorilla trichotomy in favor of a more recent association of chimpanzees and humans.

Goldman, D.; Giri, P.R.; O'Brien, J.O.



Quantitative Characterization of Protein Associations in Highly Concentrated Solution  

NASA Astrophysics Data System (ADS)

With few exceptions, one cannot reliably predict the behavior of a protein at high concentration on the basis of knowledge obtained from experiments carried out at low concentration. Detection and quantitative characterization of protein-protein interactions in the high concentration regime (> 50 g/L) therefore presents both experimental and theoretical challenges to the investigator. Two experimental methods devised in our laboratory specifically for this purpose are described. (1) Non-ideal tracer sedimentation equilibrium. Instrumentation and theory for measuring and interpreting the equilibrium gradient of a labeled dilute tracer protein in a solution containing an arbitrary concentration of one or more unlabeled macromolecules are outlined. The composition dependence of the equilibrium gradient of several proteins, including ribonuclease at concentrations up to 200 g/L and immunoglobulin G at concentrations up to 125 g/L, will be presented and interpreted in the context of models taking into account both equilibrium self-association, and nonspecific repulsive steric or electrostatic repulsion. (2) Non-ideal light scattering. Recently developed instrumentation and theory for rapid measurement and interpretation of the light scattering of a protein solution over a broad range of concentration are outlined. The concentration-dependent light scattering of chymotrypsin A at three different pH values at concentrations up to 60 g/L, and the concentration-dependent light scattering of two monoclonal antibodies at concentrations up to over 200 g/L in solutions of varying ionic strength, are quantitatively accounted for by models that take into account both nonideal repulsion between protein molecules and specific modes of equilibrium self-association.

Minton, Allen



Stress Responsive Proteins Are Actively Regulated during Rice (Oryza sativa) Embryogenesis as Indicated by Quantitative Proteomics Analysis  

PubMed Central

Embryogenesis is the initial step in a plant’s life, and the molecular changes that occur during embryonic development are largely unknown. To explore the relevant molecular events, we used the isobaric tags for relative and absolute quantification (iTRAQ) coupled with the shotgun proteomics technique (iTRAQ/Shotgun) to study the proteomic changes of rice embryos during embryogenesis. For the first time, a total of 2 165 unique proteins were identified in rice embryos, and the abundances of 867 proteins were actively changed based on the statistical evaluation of the quantitative MS/MS signals. The quantitative data were then confirmed using multiple reactions monitoring (MRM) and were also supported by our previous study based on two-dimensional gel electrophoresis (2 DE). Using the proteome at 6 days after pollination (DAP) as a reference, cluster analysis of these differential proteins throughout rice embryogenesis revealed that 25% were up-regulated and 75% were down-regulated. Gene Ontology (GO) analysis implicated that most of the up-regulated proteins were functionally categorized as stress responsive, mainly including heat shock-, lipid transfer-, and reactive oxygen species-related proteins. The stress-responsive proteins were thus postulated to play an important role during seed maturation.

Zi, Jin; Zhang, Jiyuan; Wang, Quanhui; Zhou, Baojin; Zhong, Junyan; Zhang, Chaoliang; Qiu, Xuemei; Wen, Bo; Zhang, Shenyan; Fu, Xiqin; Lin, Liang; Liu, Siqi



Improved Solubilization of Surface Proteins from Listeria monocytogenes for Two-dimensional Gel Electrophoresis  

Technology Transfer Automated Retrieval System (TEKTRAN)

Solubilization of bacterial surface (cell wall and membrane-associated) proteins for 2-DE is challenging, particularly in the case of Gram-positive bacteria. This is primarily due to strong protein association with the cell wall peptidoglycan and protein hydrophobicity. We solubilized surface protei...


Correlation between the charge of proteins in solution and in the gas phase investigated by protein charge ladders, capillary electrophoresis, and electrospray ionization mass spectrometry  

SciTech Connect

Charge ladders of bovine carbonic anhydrase II, hen egg-white lysozyme, and bovine pancreatic trypsin inhibitor, prepared by partial acetylation of primary amino groups on the surface of the protein, have been analyzed by capillary electrophoresis (CE) and on-line electrospray ionization mass spectrometry (ESIMS) using solution conditions that maintain the native structure of the protein. CE was used to separate the proteins that constitute the charge ladder into individual rungs-protein derivatives that have the same number of acetylated amino groups and approximately the same net charge in solution. ESI was used to produce ions i the gas phase of the proteins that constitute each rung of the charge ladder; the mass spectra of these ions were obtained and analyzed. The distributions in charge states observed in the gas phase for the groups of proteins comprising each rung of the charge ladders were narrow, consistent with the retention of a compact structure of the proteins in the gas phase, and substantially independent of the number of acetylated amino groups. The ions observed in the gas phase had surface charge densities in a relatively narrow range of {approximately}0.9--1.5 units of charge per 10{sup 3}{angstrom}{sup 2} of surface area (as estimated from crystallographic structures). These results demonstrate that the distribution of charge states for proteins produced in the gas phase by ESI do not necessarily reflect the net charge of the protein in solution or the number of amino groups on the protein.

Carbeck, J.D.; Gao, J.; Smith, R.D.; Whitesides, G.M. [Harvard Univ., Cambridge, MA (United States). Dept. of Chemistry and Chemical Biology; Severs, J.C.; Wu, Q. [Pacific Northwest National Lab., Richland, WA (United States). Environmental Molecular Science Lab.



Comparison of DIGE and post-stained gel electrophoresis with both traditional and SameSpots analysis for quantitative proteomics.  


2-DE is an important tool in quantitative proteomics. Here, we compare the deep purple (DP) system with DIGE using both a traditional and the SameSpots approach to gel analysis. Missing values in the traditional approach were found to be a significant issue for both systems. SameSpots attempts to address the missing value problem. SameSpots was found to increase the proportion of low volume data for DP but not for DIGE. For all the analysis methods applied in this study, the assumptions of parametric tests were met. Analysis of the same images gave significantly lower noise with SameSpots (over traditional) for DP, but no difference for DIGE. We propose that SameSpots gave lower noise with DP due to the stabilisation of the spot area by the common spot outline, but this was not seen with DIGE due to the co-detection process which stabilises the area selected. For studies where measurement of small abundance changes is required, a cost-benefit analysis highlights that DIGE was significantly cheaper regardless of the analysis methods. For studies analysing large changes, DP with SameSpots could be an effective alternative to DIGE but this will be dependent on the biological noise of the system under investigation. PMID:18246571

Karp, Natasha A; Feret, Renata; Rubtsov, Denis V; Lilley, Kathryn S



Gluten-sensitive enteropathy in Irish setter dogs: characterisation of jejunal microvillar membrane proteins by two-dimensional electrophoresis.  


This study investigated whether gluten-sensitive enteropathy (GSE) in Irish setter dogs was associated with underlying structural abnormalities of microvillar membrane proteins. Jejunal biopsies taken from eight-month-old GSE-affected dogs reared on a normal, gluten-containing diet exhibited partial villous atrophy and contained more intra-epithelial lymphocytes than controls. The morphological abnormalities were reversed by feeding a gluten-free diet for five months and the changes were accompanied by an increase in the mucosal activity of the microvillar hydrolases, particularly aminopeptidase N and dipeptidyl aminopeptidase IV, which reverted to pre-treatment levels after a gluten challenge. Two-dimensional electrophoresis of microvillar membrane proteins isolated from GSE-affected dogs revealed an essentially normal protein map that was comparable to controls. The exception was an intense 85 kDa protein spot that diminished when the affected dogs were fed a gluten-free diet and re-intensified after a gluten challenge. PMID:9243723

Pemberton, P W; Lobley, R W; Holmes, R; Sørensen, S H; Batt, R M


A novel, post-column micro-membrane reactor for fluorescent analysis of protein in capillary electrophoresis.  


Based on the semipermeability of hollow fiber membranes, a post-column membrane reactor was developed for capillary electrophoresis (CE)-laser induced fluorescence (LIF) analysis of proteins by using a hollow fiber membrane to connect the separation and detection capillaries. The membrane length between the separation and detection capillaries was 1 mm. Driven by the chemical potential difference between the separation buffer inside the membrane and the fluorescence derivatization solution outside the membrane, the derivatization reagent can be easily drawn into hollow fiber membrane to react with proteins. Also, the separation buffer can be adjusted by the derivatization solution to match the conditions of derivatization without sample loss. The effect of the separation buffer on the derivatization reaction was investigated and the results showed that even a strong acidic solution and multiple additives can be adopted in the separation buffer without destroying the post-column derivatization of proteins. Under the optimized conditions, the highly sensitive detection of BSA was achieved with a detection limit of 3.3 nmol L(-1) and a linear calibration range from 0.007 to 0.1 mg mL(-1). The proposed CE-LIF system with a post-column membrane reactor was also successfully applied to the separation and detection of proteins in rat liver and loach muscle. PMID:24015400

Liu, Fan; Zhang, Lingyi; Qian, Junhong; Ren, Jun; Gao, Fangyuan; Zhang, Weibing



Polymer microchip capillary electrophoresis of proteins either off- or on-chip labeled with chameleon dye for simplified analysis  

PubMed Central

Microchip capillary electrophoresis of proteins labeled either off- or on-chip with the “chameleon” CE dye 503 using poly(methyl methacrylate) microchips is presented. A simple dynamic coating using the cationic surfactant cetyltrimethyl ammonium bromide prevented nonspecific adsorption of protein and dye to the channel walls. The labeling reactions for both off- and on-chip labeling proceeded at room temperature without requiring heating steps. In off-chip labeling, a 9 ng/mL concentration detection limit for bovine serum albumin (BSA), corresponding to a ~7 fg (100 zmol) mass detection limit, was obtained. In on-chip tagging, the free dye and protein were placed in different reservoirs of the microchip, and an extra incubation step was not needed. A 1 ?g/mL concentration detection limit for BSA, corresponding to a ~700 fg (10 amol) mass detection limit, was obtained from this protocol. The earlier elution time of the BSA peak in on-chip labeling resulted from fewer total labels on each protein molecule. Our on-chip labeling method is an important part of automation in miniaturized devices.

Yu, Ming; Wang, Hsiang-Yu; Woolley, Adam



Inhibitor screening and selectivity assessment against multiple cellular protein kinases by capillary electrophoresis with laser-induced fluorescence detection.  


A method that can be used for screening protein kinase inhibitors (PKIs) and simultaneously assessing their selectivity is described. The method is based on simultaneously assaying multiple cellular protein kinases by performing capillary electrophoresis (CE) separation and measuring the peak areas of the phosphorylated substrate peptides. The powerful separation capability of CE combined with the highly sensitive and selective laser-induced fluorescence (LIF) detector enables the direct screening of PKIs against cell lysates, which are used as an inexpensive source of enzymes. Four cell lines, three specific substrate peptides labeled with 5-carboxyfluorescein (5-FAM), two relative specific PKIs (TBB and H-89) and one non-specific PKI (staurosporine) were utilized to prove the methodology. With this method, the inhibitory activity of the tested compounds against multiple protein kinases was identified in parallel by comparing the peak areas of the phosphorylated substrates with those obtained in the absence of any inhibitors. The reduced peak area of the phosphorylated substrate definitively represents a positive screening result. Simultaneously, assaying the inhibition of one inhibitor against mutiple cellular protein kinases enables the assessment of its selectivity. Compared to the conventional, single-target screening format, the cell lysate-based multi-target method is more informative, more straightforward and more cost effective. PMID:24164033

Zhang, Qianqian; Zhang, Xuepei; Zhang, Hanzhi; Kang, Jingwu



Effect of heat and sodium dodecyl sulfate on solubilization of proteins before two-dimensional polyacrylamide gel electrophoresis.  


To solubilize biological samples, sodium dodecyl sulfate (SDS) frequently is added and the mixture heated at 70-100 degrees C. However, two-dimensional polyacrylamide gel electrophoresis of a single protein after SDS treatment has not been reported. When rabbit-muscle creatine kinase was so run, we saw considerable difference in the gel staining pattern for the heated and nonheated enzyme dissolved in the SDS solution. After heating for 10 min at 95 degrees C the number of silver-stained spots apparent increased, and staining of several spots intensified. After 60 min, most of the discrete spots disappeared. Evidently the peptide backbone had been hydrolyzed. When the enzyme was simply left at room temperature for four days, the effects were similar. Appearance of new spots and loss of spots apparently are caused by heating alone but are intensified by SDS. Experiments with human serum albumin yielded similar results. PMID:6499173

Hodges, S C; Hirata, A A



Improved method for the simultaneous determination of whey proteins, caseins and para-kappa-casein in milk and dairy products by capillary electrophoresis.  


A capillary electrophoresis method for the simultaneous determination of whey proteins, caseins and their degradation products, such as para-kappa-casein, was proposed. The effect of several parameters (pH, ionic strength and concentration of urea in the electrophoresis buffer and applied voltage) on the analysis time and on the separation efficiency of the major milk proteins was studied. Using a hydrophilically coated capillary, in combination with electrophoresis buffer 0.48 M citric acid-13.6 mM citrate-4.8 M urea at pH 2.3, and a separation voltage of 25 kV, a complete separation of beta-lactoglobulin and para-kappa-casein was achieved, permitting the quantification of both components. PMID:11358252

Miralles, B; Rothbauer, V; Manso, M A; Amigo, L; Krause, I; Ramos, M



Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants.  

PubMed Central

The proteins of the major human snRNPs U1, U2, U4/U6 and U5 were characterised by two-dimensional electrophoresis, with isoelectric focussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second. With the exception of protein F, which exhibits an acidic pl value (pl = 3.3), the snRNP proteins are basic. Post-translational modification was found among the proteins associated specifically with the U1 and U2 particles. The most complex modification pattern was observed for the U1-specific 70K protein. This was found in at least 13 isoelectric variants, with pl values ranging from 6.7 to 8.7; these variants differed also in molecular weight. All of the 70K variants are phosphorylated in the cell. Thin-layer analysis of their tryptic phosphopeptides revealed that the 70K variants have four major phosphopeptides in common, in addition to which at least four additional serine residues are phosphorylated to different extents. The comparative phosphopeptide analysis shows that differential phosphorylation alone is not sufficient to explain the occurrence of the many isoelectric variants of 70K, so that the final charge of the 70K variants is determined both by phosphorylation and by other, as yet unidentified posttranslational modifications. By two-dimensional separation of snRNP proteins obtained from mouse Ehrlich ascites tumour cells, it was shown that the pattern of pl values of the mouse proteins was almost identical with the corresponding pattern for human proteins. Even the complex modification patterns of the 70K protein are identical in mouse and man, indicating that the presence in the cell of so many variants of this protein may have functional importance. The major difference between murine and human snRNP proteins is the absence of protein B' from mouse snRNPs. This suggests that the homologous protein B may be able to carry out the task of protein B'. Images

Woppmann, A; Patschinsky, T; Bringmann, P; Godt, F; Luhrmann, R



Application of high-performance capillary electrophoresis to the quantitative analysis of nicotine and profiling of other alkaloids in ATF-regulated tobacco products.  


Tobacco products regulated by the Bureau of Alcohol, Tobacco and Firearms (ATF), are classified at different excise tax rates according to the Code of Federal Regulations. These include the smoking (cigars, cigarettes, pipe tobacco and roll-your-own) and smokeless (chewing tobacco and snuff) tobacco products. The active principal components in all tobacco products belong to a class of compounds known as alkaloids. Nicotine is the major tobacco alkaloid, comprising about 98% of the total alkaloids. It is also the primary determinant of what constitutes a tobacco product from a regulatory standpoint. Nornicotine, anabasine and anatabine constitute the minor tobacco alkaloids of importance and interest to ATF. We have previously shown capillary electrophoresis (CE) to be a powerful analytical tool for monitoring nicotine in ATF-regulated products. Here we have extended those CE studies to (i) quantitate nicotine in ATF-regulated tobacco products and (ii) to characterize these different tobacco products according to their alkaloid profiles. Results from these studies will be presented. PMID:9511858

Lu, G H; Ralapati, S



Phosphate-affinity electrophoresis on a microchip for determination of protein kinase activity.  


We describe microchip-based phosphate-affinity electrophoresis (microPAE) for separation of peptides aimed at determination of kinase activity. The microPAE exploits two recently published technologies: autonomous sample injection for PDMS microchips and a phosphate-specific affinity ligand, Phos-tag. We prepared a fluorescently labeled substrate peptide, specific to human c-Src, and its phosphorylated form. We synthesized a Phos-tag-poly(dimethylacrylamide) conjugate. The conjugate and the sample solutions were autonomously injected into a PDMS-glass hybrid microchip. The two solutions were contacted together in the microchannel. When the peptides were electrophoresed into the Phos-tag-poly(dimethylacrylamide) region, the phosphorylated peptide was specifically trapped, and separated from the nonphosphorylated peptide in 10 s. The results were quantified by the areas of the fluorescence peaks. The calibration plot obtained with standard samples showed an excellent linearity and a LOD of 0.9% phosphorylated peptide among the total peptides. For c-Src-reacted samples, the results from the microPAE were in good agreement with those from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The microPAE was also successful in the presence of inhibitors for c-Src. The measured 50% inhibitory concentration values for staurosporine, PP2, and SU6656 were in good agreement with the literature values. PMID:19784951

Han, Aishan; Hosokawa, Kazuo; Maeda, Mizuo



Quantitative and qualitative differences in protein expression between papillary thyroid carcinoma and normal thyroid tissue  

Microsoft Academic Search

In order to better understand basic mechanisms of tumor development and identify potential new biomarkers, we have performed difference gel electrophoresis (DIGE) and peptide mass fingerprinting on pooled protein extracts from patients with papillary thyroid carcinoma (PTC) compared with matched normal thyroid tissue. Image analysis of DIGE gels comparing PTC and matched normal thyroid tissue protein indicated that 25% of

Lewis M. Brown; Steve M. Helmke; Stephen W. Hunsucker; Romana T. Netea-Maier; Simon A. Chiang; David E. Heinz; Kenneth R. Shroyer; Mark W. Duncan; Bryan R. Haugen



Enzyme and storage protein electrophoresis in varietal identification of sugar beet  

Microsoft Academic Search

Different methods of classification, based on total protein patterns as well as on specific isoenzyme patterns, were compared in order to establish an identification system for sugar beet varieties and lines. Single seed patterns and bulk extractions of total and fractionated proteins were compared on SDS-PAGE. Due to important intra-populational variation contrasting with similarity at the varietal level no method

M. Oleo; J. P. C. Geyt; M. Jacobs



Major proteins related to chlortetracycline biosynthesis in a Streptomyces aureofaciens production strain studied by quantitative proteomics  

Microsoft Academic Search

Changes in synthesis and abundance of proteins associated with chlortetracycline (CTC) production in Streptomyces aureofaciens were investigated by two-dimensional polyacrylamide gel electrophoresis of proteins pulse-labelled in vivo with L-[ 35S]methionine. Eleven individual protein spots were selected as being related to formation of the antibiotic. Expression of these prominent proteins was not observed in the non-producing mutant; moreover, they were overexpressed

X.-M. Li; J. Novotná; J. Vohradský; J. Weiser



An attempt to resolve all the various proteins in a single human cell type by two-dimensional electrophoresis: I. Extraction of all cell proteins.  


A concept is presented for estimation of the total number of different proteins in a single human cell type (exemplified here by Hep cells) by use of two-dimensional electrophoresis (2DE). This concept includes three problems, the first, investigated in this study, being the transfer of all protein species of the cells into a sample useful for separation by 2DE. Five different extraction media containing--in various combinations--urea, Nonidet P-40, Zwittergent, mercaptoethanol, dithiothreitol, and sodium dodecyl sulfate were used step by step in three different extraction procedures to extract the cell proteins. The amount of radiolabeled proteins in each extract was measured. Each extract was subjected to 2DE. From the total mass of cell proteins, 99.99% could be extracted in two steps: 96% were extracted with urea/beta-mercaptoethanol solution, the remaining 4% with sodium dodecyl sulfate/urea/beta-mercaptoethanol solution. A special class of proteins assumed to be present in the latter fraction was not detected. Thus this fraction can be omitted from the further analysis of all cell proteins by 2DE. Protein classes that possibly remain undetected by the described extraction procedures are mentioned. PMID:6499175

Klose, J; Zeindl, E



Analysis of differentially expressed mitochondrial proteins in chromophobe renal cell carcinomas and renal oncocytomas by 2-D gel electrophoresis  

PubMed Central

Renal oncocytomas (RO) and chromophobe renal cell carcinomas (RCC) display morphological and functional alterations of the mitochondria. Previous studies showed that accumulation of mitochondria in ROs is associated with somatic mutations of mitochondrial DNA (mtDNA) resulting in decreased activity of the respiratory chain complex I, whereas in chromophobe RCC only heteroplasmic mtDNA mutations were found. To identify proteins associated with these changes, for the first time we have compared the mitochondrial proteomes of mitochondria isolated from ROs and chromophobe RCCs as well as from normal kidney tissues by two-dimensional polyacrylamide gel electrophoresis. The proteome profiles were reproducible within the same group of tissues in subsequent experiments. The expression patterns within each group of samples were compared and 81 in-gel digested spots were subjected to nanoLC-MS/MS-based identification of proteins. Although the list of mitochondrial proteins identified in this study is incomplete, we identified the downregulation of NDUFS3 from complex I of the respiratory chain and upregulation of COX5A, COX5B, and ATP5H from complex IV and V in ROs. In chromophobe RCCs downregulation of ATP5A1, the alpha subunit of complex V, has been observed, but no changes in expression of other complexes of the respiratory chain were detected. To confirm the role of respiratory chain complex alterations in the morphological and/or functional changes in chromophobe RCCs and ROs, further studies will be necessary.

Yusenko, Maria V.; Ruppert, Thomas; Kovacs, Gyula



Immunoreactive Coxiella burnetii Nine Mile proteins separated by 2D electrophoresis and identified by tandem mass spectrometry  

PubMed Central

Coxiella burnetii is a Gram-negative obligate intracellular pathogen and the causative agent of Q fever in humans. Q fever causes acute flu-like symptoms and may develop into a chronic disease leading to endocarditis. Its potential as a bioweapon has led to its classification as a category B select agent. An effective inactivated whole-cell vaccine (WCV) currently exists but causes severe granulomatous/necrotizing reactions in individuals with prior exposure, and is not licensed for use in most countries. Current efforts to reduce or eliminate the deleterious reactions associated with WCVs have focused on identifying potential subunit vaccine candidates. Both humoral and T cell-mediated responses are required for protection in animal models. In this study, nine novel immunogenic C. burnetii proteins were identified in extracted whole-cell lysates using 2D electrophoresis, immunoblotting with immune guinea pig sera, and tandem MS. The immunogenic C. burnetii proteins elicited antigen-specific IgG in guinea pigs vaccinated with whole-cell killed Nine Mile phase I vaccine, suggesting a T cell-dependent response. Eleven additional proteins previously shown to react with immune human sera were also antigenic in guinea pigs, showing the relevance of the guinea pig immunization model for antigen discovery. The antigens described here warrant further investigation to validate their potential use as subunit vaccine candidates.

Deringer, James R.; Chen, Chen; Samuel, James E.; Brown, Wendy C.



Back to the basics: Maximizing the information obtained by quantitative two dimensional gel electrophoresis analyses by an appropriate experimental design and statistical analyses.  


Two dimensional gel electrophoresis has been one of the techniques most used for protein separation in proteomics experiments and still continues to be so for some species such as plants. Despite the constant technical advances and continuous improvements in the field of 2-DE, the experimental design and analysis of protein abundance data continue to be ignored or not properly documented in the literature. An appropriate experimental design, followed by decisive statistical methods is mandatory to extract all the information that is concealed in the complexity of 2-DE data. In this work we review, in a biologist's language, all the experimental design and statistical tests to be considered while planning a 2-DE based proteomics experiment and for the correct analysis and interpretation of the data. We aim to provide the researcher with an up to date introduction to these areas, starting with the experimental design and ending with the application of multivariate statistical methodologies such as PCA, ICA or neural network-based self-organizing maps. In between we have described, in an understandable way, the current methodologies available to deal with all the stages of the experimental design, data processing and analysis. PMID:20656082

Valledor, Luis; Jorrín, Jesús



Study of extraction procedures for protein analysis in plankton samples by OFFGEL electrophoresis hyphenated with Lab-on-a-chip technology.  


Extraction procedures for protein analysis from plankton samples were studied. OFFGEL electrophoresis combined with Lab-on-a-chip technology has been applied for protein analysis in plankton samples. BCR-414 (plankton) certified reference material from the European Commission was used to evaluate the protein extraction procedures. Three protein extraction procedures were studied: (1) by using Tris-HCl buffer containing a protease inhibitor cocktail, (2) urea/triton X-100 buffer extraction, and (3) using the phenol/sodium dodecyl sulphate method after different washing steps with 10% trichloroacetic acid/acetone solution and methanol. The pellet of proteins obtained was dried and then dissolved in the OFFGEL buffer. Proteins were separated according to their isoelectric points by OFFGEL electrophoresis. This separation was performed using 24cm, pH 3-10 IPG Dry Strips. The proteins present in each liquid fraction (24 fractions) were separated according to their molecular weight using a microfluidic Lab-on-a-chip electrophoresis with the Protein 80 LabChip kit. This kit allows for the separation of proteins with a molecular weight ranging from 5 to 80kDa. Taking into account the intensity and the number of the protein bands obtained, the protein extraction procedure using the phenol/sodium dodecyl sulphate after different wash steps with 10% trichloroacetic acid/acetone solution was selected. The developed method was applied for protein determination in a fresh marine plankton sample. The proteins found in this sample have a molecular weight ranging from 6.4 to 57.3kDa, and the proteins with highest molecular weight were in the OFFGEL fractions with an isoelectric point ranging from 4.40 to 8.60. The concentration of proteins were calculated using external calibration with Bovine Serum Albumin, and the protein concentrations varied from 50.0 to 925.9ngµL(-1). PMID:24054642

García-Otero, Natalia; Barciela-Alonso, Ma Carmen; Moreda-Piñeiro, Antonio; Bermejo-Barrera, Pilar



Quantitative evaluation of protein conformation in pharmaceuticals using cross-linking reactions coupled with LC-MS/MS analysis.  


The need for a simple and high-throughput method for identifying the tertiary structure of protein pharmaceuticals has increased. In this study, a simple method for mapping the protein fold is proposed for use as a complementary quality test. This method is based on cross-linking a protein using a [bis(sulfosuccinimidyl)suberate (BS(3))], followed by peptide mapping by LC-MS. Consensus interferon (CIFN) was used as the model protein. The tryptic map obtained via liquid chromatography tandem mass spectroscopy (LC-MS/MS) and the mass mapping obtained via matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy were used to identify cross-linked peptides. While LC-MS/MS analyses found that BS(3) formed cross-links in the loop region of the protein, which was regarded as the biologically active site, sodium dodecyl-sulfate polyacrylamide gel electrophoresis demonstrated that cross-linking occurred within a protein molecule, but not between protein molecules. The occurrence of cross-links at the active site depends greatly on the conformation of the protein, which is determined by the denaturing conditions. Quantitative evaluation of the tertiary structure of CIFN was thus possible by monitoring the amounts of cross-linked peptides generated. Assuming that background information is available at the development stage, this method may be applicable to process development as a complementary test for quality control. PMID:21367553

Yamaguchi, Hideto; Hirakura, Yutaka; Shirai, Hiroki; Mimura, Hisashi; Toyo'oka, Toshimasa



Microfluidic Electrophoresis Assays for Rapid Characterization of Protein in Research and Development  

NSDL National Science Digital Library

This presentation discussed the use of microfluidic-CE platforms for characterization assays such as purity assessment of monoclonal antibodies under reducing and nonreducing conditions, N-glycan profiling, and determination of protein charge heterogeneity.

Sean Sanders (Science/AAAS;); Bahram Fathollahi (PerkinElmer;); Timothy Blanc (ImClone Systems;); Joey Studts (Boehringer Ingelheim Pharma GmbH & Co. KG;)



Human cellular protein patterns and their link to genome DNA sequence data: Usefulness of two-dimensional gel electrophoresis and microsequencing  

SciTech Connect

Analysis of cellular protein patterns by computer-aided 2-dimensional gel electrophoresis together with recent advances in protein sequence analysis have made possible the establishment of comprehensive 2-dimensional gel protein databases that may link protein and DNA information and that offer a global approach offered by 2-dimensional gel protein databases it is now possible to reveal phenotype specific protein (or proteins), to microsequence them, to search for homology with previously identified proteins, to clone the cDNAs, to assign partial protein sequence to genes for which the full DNA sequence and the chromosome location is known, and to study the regulatory properties and function of groups of proteins that are coordinately expressed in a given biological process. Human 2-dimensional gel protein databases are becoming increasingly important in view of the concerted effort to map and sequence the entire genome.

Celis, J.E.; Rasmussen, H.H.; Leffers, H.; Madsen, P.; Honore, B.; Gesser, B.; Dejgaard, K. (Aarhus Univ. (Denmark)); Vandekerckhove, J. (Rijksuniversiteit Gent (Belgium))



Sub-visible particle quantitation in protein therapeutics.  


Biologics represent a large and growing segment of the therapeutic medicinal market. Sub-visible particles present in these products are a product quality attribute and a potential patient safety concern yet to be fully explored. Early and consistent particle quantitation and control throughout the product life cycle of these drugs from development to commercial lot release is critical in mitigating any concerns. This requires appropriate analytical methods which can be applied to biopharmaceuticals across a large variety of protein concentrations and modes of administration. The compendial light obscuration method for quantitating sub-visible particles in small volume parenterals is not ideally suited for therapeutic biologics. Approaches to modify the current compendial method so that it is applicable to biologics, including appropriate sample preparation, reduced assay sample volume, increased sizing information, and development of an appropriate sampling plan, are presented in this article. Successful applications of a modified light obscuration method to therapeutic protein products are demonstrated, and a strategy to utilise complimentary methods and techniques at different phases of product development is discussed. PMID:20144454

Cao, S; Jiao, N; Jiang, Y; Mire-Sluis, A; Narhi, L O



Use of capillary electrophoresis-sodium dodecyl sulfate to monitor disulfide scrambled forms of an Fc fusion protein during purification process.  


Overexpression of recombinant Fc fusion proteins in Escherichia coli frequently results in the production of inclusion bodies that are subsequently used to produce fully functional protein by an in vitro refolding process. During the refolding step, misfolded proteins such as disulfide scrambled forms can be formed, and purification steps are used to remove these product-related impurities to produce highly purified therapeutic proteins. A variety of analytical methods are commonly used to monitor protein variants throughout the purification process. Capillary electrophoresis (CE)-based techniques are gaining popularity for such applications. In this work, we used a nonreduced capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS) method for the analysis of disulfide scrambled forms in a fusion protein. Under denatured nonreduced conditions, an extra post-shoulder peak was observed at all purification steps. Detailed characterization revealed that the peak was related to the disulfide scrambled forms and was isobaric with the correctly folded product. In addition, when sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used during the CE-SDS peak characterization, we observed that the migration order of scrambled forms is reversed on CE-SDS versus SDS-PAGE. This illustrates the importance of establishing proper correlation of these two techniques when they are used interchangeably to guide the purification process and to characterize proteins. PMID:21420378

Hapuarachchi, Suminda; Fodor, Szilan; Apostol, Izydor; Huang, Gang



A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles  

Microsoft Academic Search

It is now known that nanoparticles, when exposed to biological fluid, become coated with proteins and other biomolecules to form a `protein corona'. Recent systematic studies have identified various proteins that can make up this corona, but these nanoparticle-protein interactions are still poorly understood, and quantitative studies to characterize them are few in number. Here, we have quantitatively analysed the

Carlheinz Röcker; Matthias Pötzl; Feng Zhang; Wolfgang J. Parak; G. Ulrich Nienhaus



Low-flow sheathless capillary electrophoresis-mass spectrometry for sensitive glycoform profiling of intact pharmaceutical proteins.  


Capillary electrophoresis coupled to time-of-flight mass spectrometry (CE-TOF-MS) via a porous tip sheathless electrospray ionization (ESI) interface was studied for the characterization of pharmaceutical glycoproteins. To achieve optimal glycoform separation, background electrolytes of low pH were used in conjunction with a capillary with a neutral coating exhibiting near-zero electroosmotic flow. Crucial interfacing parameters, like ESI voltage and ESI tip-to-end plate distance, were optimized for very low flow rates (?5 nL/min) in order to attain maximum sensitivity and stable performance. Under optimal conditions, the sheathless CE-MS interface provided significantly increased ionization efficiencies for intact proteins and decreased ionization suppression leading to detection limits in the picomolar-range. Analysis of a sample of recombinant human interferon-? allowed the assignment of at least 18 glycoforms, plus a variety of deamidation, succinimide, and oxidation products, representing a considerable improvement over sheath-liquid CE-MS. The sheathless CE-MS system also proved highly suitable for the glycoprofiling of recombinant human erythropoietin, revealing 74 glycoforms in a 60-min run. In addition, oxidation and acetylation products were detected, overall resulting in assignment of more than 250 different isoforms. Semiquantitative glycoprofiles could be derived for both pharmaceutical proteins, with estimated glycoform concentrations analyzed ranging from 0.35 to 950 nM. These profiles may be very useful for quality control of biopharmaceuticals and their biosimilars. PMID:23323765

Haselberg, Rob; de Jong, Gerhardus J; Somsen, Govert W



Identification of Leguminosae gums and evaluation of carob-guar mixtures by capillary zone electrophoresis of protein extracts.  


A procedure for the extraction and capillary zone electrophoresis (CZE) separation of proteins from carob, guar and tara gums in a background electrolyte (BGE) of pH 9 containing 0.1% polyvinyl alcohol is described. The CZE protein profiles exhibit characteristic peaks for each one of the Leguminosae gums, which can be used to construct models capable of identifying samples of carob, guar and tara gums, and predicting the guar content in binary carob-guar mixtures of different geographical origin and harvested in different years. The classification and prediction models are constructed by using linear discriminant analysis (LDA) and multiple linear regression (MLR), respectively. An excellent resolution between the three categories is obtained with LDA, the model being capable of classifying samples with recognition and prediction capabilities of 100%. For MLR models constructed with carob-guar mixtures with and without a common history, the average of the calibration residuals are +/- 0.50 and +/- 0.90%, respectively (average values for the 2-20% guar range). For the later model, the detection limit was 3.2% guar (from the standard deviation of 18 mixtures with 2-4% guar, and for alpha = beta = 0.05). PMID:12179992

Ruiz-Angel, María J; Simó-Alfonso, Ernesto F; Mongay-Fernández, Carlos; Ramis-Ramos, Guillermo



Quantitative determination of protein stability and ligand binding using a green fluorescent protein reporter system.  


Information about the stability of proteins is paramount to determine their optimal storage or reaction conditions. It is also essential to determine protein stability in high-throughput when screening for new or improved functions of proteins obtained from large mutant libraries. In drug discovery programs, monitoring of ligand-induced stabilization effects can be used to identify lead compounds in high-throughput. These studies require expensive biophysical instrumentation and large quantities of purified proteins. To address these issues, we developed a new method, using GFP as a reporter system to quantify the stability of a protein and its ligand-associated stabilization effects that requires neither special equipment nor extensive purification steps. Here, GFP is fused to a protein of interest (POI) through a linker and is used as a reporter system for protein unfolding and aggregation. The three POIs used in this study include the Ter-binding protein Tus, glycerol kinase and chloramphenicol acetyl transferase. The fluorescent fusion protein is subjected to irreversible thermal denaturation leading to formation of aggregates, which are eliminated by a centrifugation step. The residual fluorescence of the soluble fraction can be directly related to the stability of the POI and can be quantitatively monitored using a fluorescence plate reader. The GFP-based stability assay (GFP-Basta) was able to identify stabilizing compounds and afforded a new quantitative method for the screening and ranking of ligands for three different proteins. These applications are particularly useful for drug discovery, directed evolution, structural and functional genomics. PMID:20454718

Moreau, Morgane J J; Morin, Isabelle; Schaeffer, Patrick M



Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography–electrospray ionization mass spectrometry and capillary electrophoresis–electrospray ionization mass spectrometry of proteins  

Microsoft Academic Search

Peptides and proteins were separated by capillary electrophoresis (CE) in fused-silica capillaries coated with an irreversibly adsorbed monolayer of derivatized polystyrene nanoparticles. Whereas phosphate buffer, pH 3.10, enabled the highly efficient separation of basic proteins with plate counts up to 1?400?000 m?1, volatile buffer components such as formic acid or acetic acid titrated with ammonia to the desired pH had

Christian G Huber; Andreas Premstaller; Gerald Kleindienst



Iterative data analysis is the key for exhaustive analysis of peptide mass fingerprints from proteins separated by two-dimensional electrophoresis  

Microsoft Academic Search

Peptide mass fingerprinting (PMF) is a powerful tool for identification of proteins separated by two-dimensional electrophoresis\\u000a (2-DE). With the increase in sensitivity of peptide mass determination it becomes obvious that even spots looking well separated\\u000a on a 2-DE gel may consist of several proteins. As a result the number of mass peaks in PMFs increased dramatically leaving\\u000a many unassigned after

Frank Schmidt; Monika Schmid; Peter R. Jungblut; Jens Mattow; Axel Facius; Klaus-Peter Pleissner



Capillary HPLC–ICP MS mapping of selenocompounds in spots obtained from the 2-D gel electrophoresis of the water-soluble protein fraction of selenized yeast  

Microsoft Academic Search

A method based on ICP collision-cell MS detection in capillary HPLC was developed to gain an insight into the purity and identity of selenium-containing proteins separated by 1-D and 2-D electrophoresis. The bands and spots obtained after the separation of water-soluble proteins in selenized yeast were digested with trypsin prior to chromatography. Selenium could be detected down to the subpicogram

Laure Tastet; Dirk Schaumlöffel; Brice Bouyssiere; Ryszard Lobinski



Mobility-based selective on-line preconcentration of proteins in capillary electrophoresis by controlling electroosmotic flow.  


A simple method to perform selective on-line preconcentration of protein samples in capillary electrophoresis (CE) is described. The selectivity, based on protein electrophoretic mobility, was achieved by controlling electroosmotic flow (EOF). A short section of dialysis hollow fiber, serving as a porous joint, was connected between two lengths of fused silica capillary. High voltage was applied separately to each capillary, and the EOF in the system was controlled independently of the local electric field intensity by controlling the total voltage drop. An equation relating the EOF with the total voltage drop was derived and evaluated experimentally. On-line preconcentration of both positively charged and negatively charged model proteins was demonstrated without using discontinuous background electrolytes, and protein analytes were concentrated by approximately 60-200-fold under various conditions. For positively charged proteins, positive voltages of the same magnitude were applied at the free ends of the connected capillaries while the porous joint was grounded. This provided a zero EOF in the system and a non-zero local electric field in each capillary to drive the positively charged analytes to the porous joint. CE separation was then initiated by switching the polarity of the high voltage over the second capillary. For negatively charged proteins, the procedure was the same except negative voltages were applied at the free ends of the capillaries. Mobility-based selective on-line preconcentration was also demonstrated with two negatively charged proteins, i.e. beta-lactoglobulin B and myoglobin. In this case, negative voltages of different values were applied at the free ends of the capillaries with different values, which provided a non-zero EOF in the system. The direction of EOF was the same as that of the electrophoretic migration velocities of the protein analytes in the first capillary and opposite in the second capillary. By controlling the EOF, beta-lactoglobulin B, which has a higher mobility, could be concentrated over 150-fold with a 15 min injection while myoglobin, which has a lower mobility, was eliminated from the system. PMID:14753681

Wang, Qinggang; Yue, Bingfang; Lee, Milton L



Imaging metals in proteins by combining electrophoresis with rapid x-ray fluorescence mapping.  

SciTech Connect

Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal?protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis.

Finney, L.; Chishti, Y.; Khare, T.; Giometti, C.; Levina, A.; Lay, P. A.; Vogt, S.; Univ. of Sydney; Northwestern Univ.



An integrated PCR microfluidic chip incorporating aseptic electrochemical cell lysis and capillary electrophoresis amperometric DNA detection for rapid and quantitative genetic analysis.  


A fully integrated microchip for performing cell lysis, polymerase chain reaction (PCR) and quantitative analysis of DNA amplicons in a single step is described herein. The chip was built on glass substrate using an indium-tin-oxide (ITO) microheater and PDMS engraved microchannels, which integrated an electrochemical cell lysis zone, a continuous flow PCR module and capillary electrophoresis amperometric detection (CE-AD) system. The total length of the microchannel was 4625 mm for performing 25 cycles of flow-through PCR and was laid on a handheld form factor of 96 × 96 mm(2) area. The key to the fabrication of such a device lies in the use of a single medium to carry out different kinds of biochemical reactions and hence, a reagentless electrochemical cell lysis protocol was integrated on the microchip which was capable of lysing most cell types, including difficult to lyse gram positive bacteria. The lysate contained genomic DNA from a sample which was proven to be suitable for PCR reactions. Genetic analysis was successfully performed on the microchip with purified lambda phage genomic DNA and various cell types, including non-tumorigenic MCF-10A and tumorigenic MCF-7 human cell lines, gram negative bacteria Escherichia coli O157:H7, and gram positive bacteria Bacillus subtilis, at an optimized flow rate of 5 ?l min(-1). For the detection of amplicon DNA, a CE-AD system was used, with semisolid alkaline agarose within the capillary microchannel to minimize interference from cell debris and for efficient resolution of DNA fragments. High signal to noise ratio during amperometric detection and the use of online FFT filtering protocol enhanced the limit of detection of DNA amplicons. Therefore, with a combination of portability, cost-effectiveness and performance, the proposed integrated PCR microchip can be used for one step genetic analysis of most of the cell types and will enable more accessible healthcare. PMID:22960653

Jha, Sandeep Kumar; Chand, Rohit; Han, Dawoon; Jang, You-Cheol; Ra, Gyu-Sik; Kim, Joung Sug; Nahm, Baek-Hie; Kim, Yong-Sang



21 CFR 862.2485 - Electrophoresis apparatus for clinical use.  

Code of Federal Regulations, 2010 CFR

...2010-04-01 false Electrophoresis apparatus for clinical...Instruments § 862.2485 Electrophoresis apparatus for clinical...Identification. An electrophoresis apparatus for clinical...including plasma proteins, lipoproteins, enzymes, and...



21 CFR 862.2485 - Electrophoresis apparatus for clinical use.  

Code of Federal Regulations, 2010 CFR

...2009-04-01 false Electrophoresis apparatus for clinical...Instruments § 862.2485 Electrophoresis apparatus for clinical...Identification. An electrophoresis apparatus for clinical...including plasma proteins, lipoproteins, enzymes, and...



21 CFR 862.2485 - Electrophoresis apparatus for clinical use.  

Code of Federal Regulations, 2013 CFR

...Electrophoresis apparatus for clinical use. 862.2485 Section 862...Electrophoresis apparatus for clinical use. (a) Identification. ...electrophoresis apparatus for clinical use is a device intended to separate...plasma proteins, lipoproteins, enzymes, and hemoglobulins on...



Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their relation with semen freezability.  


The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. Of the 25 proteins identified, two spots had greater relative content (P < 0.05) in seminal plasma samples collected from stallions with high semen freezability: spot 5 (80-85 kDa, isoelectric point [pI] 7.54), identified as CRISP-3; and spot 45 (18.2 kDa, pI 5.0-5.2), identified as HSP-2. Conversely, protein content was greater (P < 0.05) in seminal plasma samples from stallions with low semen freezability: spot 7 (75.4 kDa, pI 6.9-7.4), identified as lactoferrin; spot 15 (26.7 kDa, pI 5.51), identified as kallikrein; spot 25 (25 kDa, pI 7.54), identified as CRISP-3; and spot 35 (13.9 kDa, pI 3.8-4.2), identified as HSP-1. In conclusion, there were differences in the seminal plasma protein profile from stallions with high and low semen freezability. Furthermore, CRISP-3 and HSP-2 were potential seminal plasma markers of high semen freezability. PMID:21601917

Jobim, M I M; Trein, C; Zirkler, H; Gregory, R M; Sieme, H; Mattos, R C



Determination of soya protein in meat products by standard curves obtained from SDS gel electrophoresis  

Microsoft Academic Search

Zusammenfassung Diese Methode basiert auf der densitometrischen Auswertung eines Dodecylsulfat-Polyacrylamid-Elektrophoresegels und dient der Identifizierung von Sojaeiweiß in rohen und erhitzten Fleischwaren, die noch andere Zusätze erhalten. Die Proteine werden in Tris\\/Borsäure-Puffer (pH 8,2) mit 3% SDS and 3% 2-Mercaptoethanol aufgelöst. Die Elektrophorese wurde in 12% Polyacrylamid mit SDS, pH 9,18, durchgeführt. Die qualitative Bestimmung ist sowohl in rohen als in

Else Molander



Application of the copolymers containing sulfobetaine methacrylate in protein separation by capillary electrophoresis.  


This study describes the formation of highly efficient antiprotein adsorption random copolymer coating of poly(N,N-dimethylacrylamide-co-sulfobetaine methacrylate) (poly(DMA-co-SBMA)) on the fused-silica capillary inner wall. Firstly, the poly(DMA-co-SBMA)s with different feed ratio (SBMA/DMA) were synthesized via the reversible addition fragmentation chain transfer polymerization. And then, X-ray photoelectron spectroscopy (XPS) and water contact angle (CA) were used to investigate the composition and hydrophilicity of poly(DMA-co-SBMA) coating formed on the glass slide surfaces. CA measurements revealed that the poly(DMA-co-SBMA) coating became more hydrophilic with the increment of feed ratio (SBMA/DMA), and at the same time, the XPS results showed that the coating ability was also increased with the increment of feed ratio. Followed, the copolymer was applied to coat the fused-silica capillary inner wall, and the coated capillary was used to separate the mixture of proteins (lysozyme, cytochrome c, ribonuclease A, and ?-chymotrypsinogen A) in a pH range from 3.0 to 5.0. Under the optimum conditions, an excellent separation of basic proteins with peak efficiencies ranging from 551,000 to 1509,000?N/m had been accomplished within 10?min. Furthermore, the effect of coating composition on protein separation was also investigated through the comparison of separation efficiency achieved by using bare, PSBMA- and poly(DMA-co-SBMA)-coated capillary, respectively. PMID:23909655

Cao, Fuhu; Tan, Lin; Xiang, Lina; Liu, Songtao; Wang, Yanmei



A quantitative method for detection of spliced X-box binding protein-1 (XBP1) mRNA as a measure of endoplasmic reticulum (ER) stress.  


Endoplasmic reticulum (ER) stress is increasingly recognized as an important mechanism in a wide range of diseases including cystic fibrosis, alpha-1 antitrypsin deficiency, Parkinson's and Alzheimer's disease. Therefore, there is an increased need for reliable and quantitative markers for detection of ER stress in human tissues and cells. Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause ER stress, which leads to the activation of the unfolded protein response (UPR). UPR signaling involves splicing of X-box binding protein-1 (XBP1) mRNA, which is frequently used as a marker for ER stress. In most studies, the splicing of the XBP1 mRNA is visualized by gel electrophoresis which is laborious and difficult to quantify. In the present study, we have developed and validated a quantitative real-time RT-PCR method to detect the spliced form of XBP1 mRNA. PMID:22038282

van Schadewijk, Annemarie; van't Wout, Emily F A; Stolk, Jan; Hiemstra, Pieter S



Quantitative analysis of protein dynamics during asymmetric cell division.  


In dividing Drosophila sensory organ precursor (SOP) cells, the fate determinant Numb and its associated adaptor protein Pon localize asymmetrically and segregate into the anterior daughter cell, where Numb influences cell fate by repressing Notch signaling. Asymmetric localization of both proteins requires the protein kinase aPKC and its substrate Lethal (2) giant larvae (Lgl). Because both Numb and Pon localization require actin and myosin, lateral transport along the cell cortex has been proposed as a possible mechanism for their asymmetric distribution. Here, we use quantitative live analysis of GFP-Pon and Numb-GFP fluorescence and fluorescence recovery after photobleaching (FRAP) to characterize the dynamics of Numb and Pon localization during SOP division. We demonstrate that Numb and Pon rapidly exchange between a cytoplasmic pool and the cell cortex and that preferential recruitment from the cytoplasm is responsible for their asymmetric distribution during mitosis. Expression of a constitutively active form of aPKC impairs membrane recruitment of GFP-Pon. This defect can be rescued by coexpression of nonphosphorylatable Lgl, indicating that Lgl is the main target of aPKC. We propose that a high-affinity binding site is asymmetrically distributed by aPKC and Lgl and is responsible for asymmetric localization of cell-fate determinants during mitosis. PMID:16243032

Mayer, Bernd; Emery, Gregory; Berdnik, Daniela; Wirtz-Peitz, Frederik; Knoblich, Juergen A



Separation of metalloproteins using a novel metal ion contaminant sweeping technique and detection of protein-bound copper by a metal ion probe in polyacrylamide gel electrophoresis: distribution of copper in human serum.  


A polyacrylamide gel electrophoresis (PAGE)-based method has been developed, consisting of two types of gel electrophoresis, to obtain an accurate distribution of protein-bound metal ions in biological samples. First, proteins are separated by PAGE without the uptake of contaminant metal ions in the separation field and dissociation of metal ions from the proteins. This is followed by another PAGE for the separation and detection of protein-bound metal ions in small volume samples with high sensitivity in the ppt range using a fluorescent metal probe. The former is a new technique using blue-native (BN) PAGE to electrophoretically sweep all metal contaminants by employing two kinds of chelating agents. These agents form complexes with contaminants in the gel and the separation buffer solution, which migrate towards opposite pole directions, thus lowering the contaminants to below the ppt level during separation. This is termed "Metal Ion Contaminant Sweeping BN-PAGE (MICS-BN-PAGE)". After the separation of proteins under these first metal-free conditions, the metal ions in the gel fractions are eluted, followed by derivatization of copper ions into the metal probe complexes to be separated and determined by fluorescence detection in the second PAGE. In this PAGE-based method, the copper ions bound to ceruloplasmin and superoxide dismutase were quantitatively determined, in addition to the exchangeable albumin-bound copper ions. This system successfully provided distribution maps of protein-copper in human serum. The precise distribution of copper in human serum was investigated, and found to be different from that which is widely accepted. PMID:23964357

Saito, Shingo; Kawashima, Mitsuyoshi; Ohshima, Hiroki; Enomoto, Kazuki; Sato, Makoto; Yoshimura, Hajime; Yoshimoto, Keitaro; Maeda, Mizuo; Shibukawa, Masami



Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models.  


The Bradford assay is a simple method for protein quantitation, but variation in the results between proteins is a matter of concern. In this study, we compared and normalized quantitative values from two models for protein quantitation, where the residues in the protein that bind to anionic Coomassie Brilliant Blue G-250 comprise either Arg and Lys (Method 1, M1) or Arg, Lys, and His (Method 2, M2). Use of the M2 model yielded much more consistent quantitation values compared with use of the M1 model, which exhibited marked overestimations against protein standards. PMID:23201266

Ku, Hyung-Keun; Lim, Hyuk-Min; Oh, Kyong-Hwa; Yang, Hyo-Jin; Jeong, Ji-Seon; Kim, Sook-Kyung



Quantitative recovery of radioactivity in automated protein sequencing cycles  

SciTech Connect

When working with radioactive samples, it is sometimes necessary to determine which amino acid residue has a radioactive label. The Applied Biosystems model 470A protein sequencer has been modified so that 40% of the extracted PTH amino acids at each cycle are automatically injected into an Applied Biosystems model 120A PTH Analyzer, while the remaining 60% are quantitatively recovered in the fraction collector. This was accomplished by altering the tubing connections between the auto-conversion flask, the high pressure sample injection valve and the fraction collector, and by modifying some of the steps in the Sequencer and PTH analyzer programs. Details of these changes will be given, as well as illustrations, using actual data obtained from sequencing a radiolabeled peptide.

Fields, R.; Stein, S.



Free-Flow Zone Electrophoresis of Peptides and Proteins in PDMS Microchip for Narrow pI Range Sample Prefractionation Coupled with Mass Spectrometry  

PubMed Central

In this paper, we are evaluating the strategy of sorting peptides / proteins based on the charge to mass without resorting to ampholytes and / or isoelectric focusing, using a single- and two-step free-flow zone electrophoresis. We developed a simple fabrication method to create a salt bridge for free-flow zone electrophoresis in PDMS chips by surface printing a hydrophobic layer on a glass substrate. Since the surface-printed hydrophobic layer prevents plasma bonding between the PDMS chip and the substrate, an electrical junction gap can be created for free-flow zone electrophoresis. With this device, we demonstrated a separation of positive and negative peptides and proteins at a given pH in standard buffer systems, and validated the sorting result with LC/MS. Furthermore, we coupled two sorting steps via off-chip titration, and isolated peptides within specific pI ranges from sample mixtures, where the pI range was simply set by the pH values of the buffer solutions. This free-flow zone electrophoresis sorting device, with its simplicity of fabrication, and a sorting resolution of 0.5 pH unit, can potentially be a high-throughput sample fractionation tool for targeted proteomics using LC/MS.

Song, Yong-Ak; Chan, Michael; Celio, Chris; Tannenbaum, Steven R.; Wishnok, John S.; Han, Jongyoon



Quantitative Characterization of Local Protein Solvation To Predict Solvent Effects on Protein Structure  

PubMed Central

Characterization of solvent preferences of proteins is essential to the understanding of solvent effects on protein structure and stability. Although it is generally believed that solvent preferences at distinct loci of a protein surface may differ, quantitative characterization of local protein solvation has remained elusive. In this study, we show that local solvation preferences can be quantified over the entire protein surface from extended molecular dynamics simulations. By subjecting microsecond trajectories of two proteins (lysozyme and antibody fragment D1.3) in 4 M glycerol to rigorous statistical analyses, solvent preferences of individual protein residues are quantified by local preferential interaction coefficients. Local solvent preferences for glycerol vary widely from residue to residue and may change as a result of protein side-chain motions that are slower than the longest intrinsic solvation timescale of ?10 ns. Differences of local solvent preferences between distinct protein side-chain conformations predict solvent effects on local protein structure in good agreement with experiment. This study extends the application scope of preferential interaction theory and enables molecular understanding of solvent effects on protein structure through comprehensive characterization of local protein solvation.

Vagenende, Vincent; Trout, Bernhardt L.



Electrophoresis of tear proteins as a new diagnostic tool for two high risk groups for dry eye: computer users and contact lens wearers  

PubMed Central

Rationale: Dry eye is the most prevalent condition seen by the ophthalmologist, in particular in elderly. The identification of new common risk factors (computer use and contact lens wear) extends the disease among the young people. The early diagnosis of dry eye is essential, but difficult, because the biochemical changes in tear film usually occur before any detectable signs. Due its advantages, electrophoresis of tear proteins could be an important tool for diagnosis of tear film impairment in high risk groups for dry eye. Objective: The role of tear proteins electrophoresis in early diagnosis of dry eye related to computer use and contact lens wear, as well as the biochemical changes in these high risk groups are presented. Methods: This review will summarize the actual data concerning the electrophoretic changes of tear proteins in computer users and contact lens wearers, two common high risk groups for dry eye. Discussion: Electrophoresis of tear proteins using automated system Hyrys–Hydrasys SEBIA France is an important tool for early diagnosis of tear film alterations and monitoring of therapy. The quantification of many proteins in a single analysis using a small quantity of unconcentrated reflex tears is the main advantage of this technique. Electrophoresis of tear proteins should became a prerequisite, in particular for computer users less than 3h/day, as well as at prescribing contact lenses. Abbreviations: DED– dry eye disease, EGF–epidermal growth factor, IL interleukins, MMP–metalloproteinase, ELISA– Enzyme–linked immunosorbent assay, SDS– sodium dodecyl sulfate, CVS– computer vision syndrome, CLRDE– contact lens– related dry eye



Comparison of two-dimensional electrophoresis patterns of heat shock protein Hsp27 species in normal and cardiomyopathic hearts.  


Heat shock protein Hsp27 occurs in a complex pattern in human myocardial tissue. Normal and failing explanted human heart from patients with dilated cardiomyopathy (DCM) or ischemic heart failure (IHF), respectively, were analyzed by high resolution two-dimensional electrophoresis (23x30 cm) and Hsp27 immunostaining. Twelve Hsp27 spots in DCM samples were significantly altered in intensity and ten of these were significantly changed in IHF. Four spots (h1, h2, h4, h5) in DCM samples and three spots (h2, h4, h5) in IHF at a molecular mass of 28 kDa were decreased in intensity. In this study, investigating left ventricles of human myocardium, spot h4 was only detected in normal heart samples. On the other hand, spots with a lower molecular mass of 27 kDa (h14, h15, h17, h20, h21) and 22-23 kDa (46, h47, h50) increased in intensity in failing hearts, suggesting that some form of Hsp27 degradation occurs during heart failure. PMID:10612289

Scheler, C; Li, X P; Salnikow, J; Dunn, M J; Jungblut, P R



Simulating Electrophoresis.  

ERIC Educational Resources Information Center

|Describes a DNA fingerprinting simulation that uses vegetable food coloring and plastic food containers instead of DNA and expensive gel electrophoresis chambers. Allows students to decipher unknown combinations of dyes in a method similar to that used to decipher samples of DNA in DNA fingerprint techniques. (JRH)|

Moertel, Cheryl; Frutiger, Bruce



Noninvasive quantitative imaging of protein-protein interactions in living subjects  

PubMed Central

We are developing methods to image molecular and cellular events in living subjects. In this study, we validate imaging of protein—protein interactions in living mice by using bioluminescent optical imaging. We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible. We employ the NF-?B promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID). We modulate the NF-?B promoter through tumor necrosis factor ?. Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy. We demonstrate the ability to detect this induced protein–protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells. The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein–protein interactions in living subjects. The approaches validated should have important implications for the study of protein–protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein–protein interactions.

Ray, P.; Pimenta, H.; Paulmurugan, R.; Berger, F.; Phelps, M. E.; Iyer, M.; Gambhir, S. S.



Segmented field OFFGEL® electrophoresis.  


A multielectrode setup for protein OFFGEL electrophoresis that significantly improves protein separation efficiency has been developed. Here, the electric field is applied by segments between seven electrodes connected in series to six independent power supplies. The aim of this strategy is to distribute evenly the electric field along the multiwell system, and as a consequence to enhance electrophoresis in terms of separation time, resolution, and protein collection efficiency, while minimizing the overall potential difference and therefore the Joule heating. The performances were compared to a standard two-electrode setup for OFFGEL fractionation of a protein mixture, using UV-Vis spectroscopy for quantification and MALDI-MS for identification. The electrophoretic separation process was simulated, and optimized by solving the time-dependent Nernst-Planck differential equation. PMID:23086720

Tobolkina, Elena; Cortés-Salazar, Fernando; Momotenko, Dmitry; Maillard, Julien; Girault, Hubert H



Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli.  

PubMed Central

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in the terminal stages of bacterial peptidoglycan assembly. As their name implies, these proteins also covalently bind and are inhibited by beta-lactam antibiotics. Although many studies have examined the relative binding affinities of a number of beta-lactam antibiotics, a surprisingly small number of studies have addressed the absolute numbers of each of the PBPs present in the bacterial cell. In the present study, the PBP values initially reported in Escherichia coli almost 20 years ago by B. G. Spratt (Eur. J. Biochem. 72:341-352, 1977) were refined. The individual PBPs from a known number of bacteria radiolabeled with [3H]benzylpenicillin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactive bands were located, excised, and quantitatively extracted from the gel slices. The radioactivity was measured by scintillation counting, and the absolute disintegrations per minute were calculated. From the specific activity of the labeled penicillin, the absolute disintegrations per minute, and the CFU per milliliter, a determination of the number of each of the PBPs per cell was made. The measurements were performed on multiple samples to place statistical limits on the numbers obtained. The values for the individual PBPs found in E. coli deviated in several ways from the previously reported observations. Of particular significance is the higher number of molecules of PBP 2 and 3 observed, since these PBPs are known to participate in cell morphogenesis. The PBP content in both rich Luria broth medium and M9 minimal medium was determined, with the slower-growing cells in minimal medium possessing fewer of the individual PBPs per cell.

Dougherty, T J; Kennedy, K; Kessler, R E; Pucci, M J



A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry.  


Protein complexes form, dissociate and re-form in order to perform specific cellular functions. In this two-pronged protocol, noncovalent protein complexes are initially isolated by affinity purification for subsequent identification of the components by liquid chromatography high-resolution mass spectrometry (LC-MS) on a hybrid LTQ Orbitrap Velos. In the second prong of the approach, the affinity-purification strategy includes a chemical cross-linking step to 'freeze' a series of concurrently formed, heterogeneous protein subcomplex species that are visualized by gel electrophoresis. This branch of the methodology amalgamates standard and well-practiced laboratory methods to reveal compositional changes that occur in protein complex architecture. By using mouse N-terminally tagged streptavidin-binding peptide-hemagglutinin-TANK-binding kinase 1 (SH-TBK1), we chemically cross-linked the affinity-purified complex of SH-TBK1 with the homobifunctional lysine-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), and we separated the resultant protein complexes by denaturation and by silver-stained one- and two-dimensional SDS-PAGE. We observed a range of cross-linked TBK1 complexes of variable pI and M(r) and confirmed them by immunoblotting. LC-MS analysis of in situ-digested cross-linked proteins shows differences in the composition of the TBK1 subcomplexes. The protocol is inherently simple and can be readily extended to the investigation of a range of protein complexes. From cell lysis to data generation by LC-MS, the protocol takes approximately 2.5 to 5.5 d to perform. PMID:23237831

Rudashevskaya, Elena L; Sacco, Roberto; Kratochwill, Klaus; Huber, Marie L; Gstaiger, Matthias; Superti-Furga, Giulio; Bennett, Keiryn L



Quantitative analysis of pheromone-binding protein specificity.  


Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins, using ?-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila odorant-binding protein that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in Escherichia?coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between ?-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the ?-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ?100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ?200?nM for the silk moth pheromone bombykol and ?90?nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the Laughlin, Ha, Jones and Smith model of pheromone reception are discussed. PMID:23121132

Katti, S; Lokhande, N; González, D; Cassill, A; Renthal, R



Electrophoresis in space.  


Programs for free flow electrophoresis in microgravity over the past 25 years are reviewed. Several studies accomplished during 20 spaceflight missions have demonstrated that sample throughput is significantly higher in microgravity than on the ground. Some studies have shown that resolution is also increased. However, many cell separation trials have fallen victim to difficulties associated with experimenting in the microgravity environment such as microbial contamination, air bubbles in electrophoresis chambers, and inadequate facilities for maintaining cells before and after separation. Recent studies suggest that the charge density of cells at their surface may also be modified in microgravity. If this result is confirmed, a further cellular mechanism of "sensing" the low gravity environment will have been found. Several free fluid electrophoresis devices are now available. Most have been tried at least once in microgravity. Newer units not yet tested in spaceflight have been designed to accommodate problems associated with space processing. The USCEPS device and the Japanese FFEU device are specifically designed for sterile operations, whereas the Octopus device is designed to reduce electroosmotic and electrohydrodynamic effects, which become dominant and detrimental in microgravity. Some of these devices will also separate proteins by zone electrophoresis, isotachophoresis, or isoelectric focusing in a single unit. Separation experiments with standard test particles are useful and necessary for testing and optimizing new space hardware. A cohesive free fluid electrophoresis program in the future will obviously require (1) flight opportunities and funding, (2) identification of suitable cellular and macromolecular candidate samples, and (3) provision of a proper interface of electrophoresis processing equipment with biotechnological facilities--equipment like bioreactors and protein crystal growth chambers. The authors feel that such capabilities will lead to the production of commercially useful quantities of target products and to an accumulation of new knowledge relating to the complexities of electrostatic phenomena at the cell surface. PMID:10660776

Bauer, J; Hymer, W C; Morrison, D R; Kobayashi, H; Seaman, G V; Weber, G



Identification of Proteins Related to Nickel Homeostasis in Helicobater pylori by Immobilized Metal Affinity Chromatography and Two-Dimensional Gel Electrophoresis.  


Helicobacter pylori (H. pylori) is a widespread human pathogen causing peptic ulcers and chronic gastritis. Maintaining nickel homeostasis is crucial for the establishment of H. pylori infection in humans. We used immobilized-nickel affinity chromatography to isolate Ni-related proteins from H. pylori cell extracts. Two-dimensional gel electrophoresis and mass spectrometry were employed to separate and identify twenty two Ni-interacting proteins in H. pylori. These Ni-interacting proteins can be classified into several general functional categories, including cellular processes (HspA, HspB, TsaA, and NapA), enzymes (Urease, Fumarase, GuaB, Cad, PPase, and DmpI), membrane-associated proteins (OM jhp1427 and HpaA), iron storage protein (Pfr), and hypothetical proteins (HP0271, HP jhp0216, HP jhp0301, HP0721, HP0614, and HP jhp0118). The implication of these proteins in nickel homeostasis is discussed. PMID:18288244

Sun, Xuesong; Ge, Ruiguang; Chiu, Jen-Fu; Sun, Hongzhe; He, Qing-Yu



Chicken liver TGGCA protein purified by preparative mobility shift electrophoresis (PMSE) shows a 36.8 to 29.8 kd microheterogeneity.  

PubMed Central

The TGGCA protein, the chicken homologue of HeLa cell NF-I, was purified to homogeneity from liver tissue by a procedure which includes preparative mobility shift electrophoresis (PMSE) as the final step. PMSE was here adjusted for the isolation of the TGGCA protein, but can be used as a general method to characterize the protein moiety of specific DNA-binding proteins. The TGGCA protein is a family of 6 protein species, which show minor differences in molecular weight from 36.8kd to 29.8kd. This microheterogeneity differs from the size distribution reported for HeLa cell NF-I polypeptides. All species of the TGGCA protein bind identically to a synthetic DNA-binding site and appear to be highly related in primary structure. We discuss the possible functional importance of this microheterogeneity. Images

Rupp, R A; Sippel, A E



Capillary electrophoresis with laser-induced fluorescence detection of proteins from two types of complex sample matrices: food and biological fluids.  


Sample preparation and laser-induced fluorescence detection are two key steps of the analytical methodology to determine by capillary electrophoresis low concentrations of proteins in complex sample matrices. In this chapter the options of performing both steps in different ways are shown by detailing the analysis of the allergen ?-lactoglobulin in food products for infants and the analysis of the isoforms of alpha 1-acid glycoprotein, a potential biomarker, in serum and secretome. PMID:23386346

Garrido-Medina, Raul; Puerta, Angel; Pelaez-Lorenzo, Cristina; Rivera-Monroy, Zuly; Guttman, Andras; Diez-Masa, Jose Carlos; de Frutos, Mercedes



Separation and identification of hen egg protein isoforms using SDS–PAGE and 2D gel electrophoresis with MALDI-TOF mass spectrometry  

Microsoft Academic Search

Knowledge of the chemical composition and physicochemical properties of native egg white and yolk is necessary to interpret the functional and biological properties attributed to specific egg components. To date, many of the proteins located in this complex biological fluid remain uncharacterised, if not unknown. High-resolution techniques for proteome analysis, including SDS–PAGE and 2-dimensional (2D) gel electrophoresis, combined with mass

Vassilios Raikos; Rasmus Hansen; Lydia Campbell; Stephen R. Euston



Identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry  

Microsoft Academic Search

Despite improvements in treatment of patients with head and neck squamous cell carcinoma (HNSCC) over the last two decades,\\u000a the survival rate of these patients has not increased significantly. One of the major factors in the poor outcome of the disease\\u000a is regional metastasis. To better understand the mechanisms of this process at the protein level, we performed two-dimensional\\u000a electrophoresis

Weiguo Wu; Ximing Tang; Wei Hu; Reuben Lotan; Waun Ki Hong; Li Mao



Global metabolic regulation analysis for Escherichia coli K12 based on protein expression by 2-dimensional electrophoresis and enzyme activity measurement  

Microsoft Academic Search

Regulation of the main metabolic pathways of Escherichia coli K12 was investigated based on 2-dimensional electrophoresis (2DE) and the measurement of enzyme activities. The cells were grown aerobically in different carbon sources, such as glucose, acetate, gluconate or glycerol. Microaerobic cultivation was also conducted with glucose as a carbon source. Fifty-two proteins could be identified based on 2DE, and 26

L. Peng; K. Shimizu



Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing.  


A capillary electrophoresis (CE)-based enzyme assay method has been developed to screen protein kinase inhibitors. Four human kinases GSK3?, DYRK1A, CDK5/p25 and CDK1/cyclin B were chosen to test this novel method. These enzymes have been identified as very promising targets to develop treatments against cancer and neurodegenerative diseases. The efficiency of drugs against these relevant biological targets has never been carried out by CE. For this proposal, the capillary was used as a nanoreactor in which four reactants (the enzyme, its two substrates and its potential inhibitor) were successively injected, mixed by using transverse diffusion of laminar flow profiles and incubated. The adenosine 5'-diphosphate (ADP) formed during the enzymatic reaction was detected by UV and quantified. The efficiency of the developed CE method was validated by determining the IC50 values of a wide variety of inhibitors covering a large domain of affinity toward kinases and containing representative and chemically divergent skeletons. Excellent agreement was found between the results obtained by CE and those reported in the literature when using conventional radiometric enzyme assays. Moreover, CE was successfully used to determine the inhibitory effect of several potential inhibitors that was not yet assessed by conventional methods and is crucial for structure activity relation studies. This novel CE method is simple, rapid, very economic (few tens of nanoliters per IC50) and eco-friendly since no radioactivity was required. It could be extended to high-throughput screening of kinase inhibitors, which is of great interest for biomedical and pharmaceutical research fields. PMID:24075461

Nehmé, Hala; Nehmé, Reine; Lafite, Pierre; Routier, Sylvain; Morin, Philippe



Analysis of proteins copurifying with the cd4\\/lck complex using one-dimensional polyacrylamide gel electrophoresis and mass spectrometry: comparison with affinity-tag based protein detection and evaluation of different solubilization methods  

Microsoft Academic Search

Mass spectrometry-based identification of the components of affinity purified protein complexes after polyacrylamide gel electrophoresis\\u000a (PAGE) and in-gel digest has become very popular for the detection of novel protein interactions. As an alternative, the entire\\u000a protein complex can be subjected to proteolytic cleavage followed by chromatographic separation of the peptides. Based on\\u000a our earlier report of a method using affinity

Oliver K. Bernhard; Anthony L. Cunningham; Margaret M. Sheil



Preparative isoelectric focusing and preparative electrophoresis of hydrophobic Candida albicans cell wall proteins with in-line transfer to polyvinylidene difluoride membranes for sequencing.  


Hydrophobic proteins in the cell wall of the opportunistic fungal pathogen Candida albicans are involved in adhesion of this organism to host tissue and thus play a role in its pathogenicity. The hydrophobic nature of these proteins results in their loss during purification due to adsorption to apparatus surfaces. This problem, combined with their low abundance, has made it problematic to purify the hydrophobic proteins in sufficient quantity for sequencing or biochemical analysis. We describe a system that combines preparative isoelectric focusing with continuous elution preparative electrophoresis. The system provides a two-dimensional protein separation while maintaining protein solubility and minimizing protein loss due to adsorption. In addition, we have added an in-line transfer of electrophoretic fractions directly to polyvinylidene difluoride (PVDF) membranes, which further reduces both exposure to apparatus surfaces and purification time. PMID:9629897

Masuoka, J; Glee, P M; Hazen, K C



Separation of olive proteins combining a simple extraction method and a selective capillary electrophoresis (CE) approach: application to raw and table olive samples.  


A simple extraction method was developed to extract proteins from olive samples based on chloroform/methanol extraction followed by a protein precipitation with cold acetone. Then, a capillary electrophoresis (CE) method was carried out using an acid buffer (1 M formic acid at pH 2) to ensure a positive net charge for proteins and a neutral charge for potential interferents as polyphenols. The method developed was applied to raw and table olive samples. Interestingly, raw olive samples showed differences in protein profiles depending upon the botanical variety of olives and their geographical region. Protein profiles obtained for table olives also showed differences according to the sample treatment. Thus, a signal reduction in the electropherograms obtained for black olives was observed in comparison to those achieved for treated green olives. In this work, the use of protein profiles was demonstrated to be a powerful tool for studying variations among olive samples. PMID:21038920

Montealegre, Cristina; Marina, Maria Luisa; García-Ruiz, Carmen



Protein Microarrays for Quantitative Detection of PAI-1 in Serum  

PubMed Central

Objective Plasminogen activator inhibitor-1 (PAI-1), one crucial component of the plasminogen activator system, is a major player in the pathogenesis of many vascular diseases as well as in cancer. High levels of PAI-1 in breast cancer tissue are associated with poor prognosis. The aim of this study is to evaluate rigorously the potential of serum PAI-1 concentration functioning as a general screening test in diagnostic or prognostic assays. Methods A protein-microarray-based sandwich fluorescence immunoassay (FIA) was developed to detect PAI-1 in serum. Several conditions of this microarray-based FIA were optimized to establish an efficacious method. Serum specimens of 84 healthy women and 285 women with breast cancer were analyzed using the optimized FIA microarray. Results The median serum PAI-1 level of breast cancer patients was higher than that of healthy women (109.7 ng/ml vs. 63.4 ng/ml). Analysis of covariance revealed that PAI-1 levels of the two groups were significantly different (P<0.001) when controlling for an age effect on PAI-1 levels. However, PAI-1 values in TNM stage I?IV patients respectively were not significantly different from each other. Conclusion This microarray-based sandwich FIA holds potential for quantitative analysis of tumor markers such as PAI-1.

Ma, Xu



Difference gel electrophoresis.  


DIGE is a protein labelling and separation technique allowing quantitative proteomics of two or more samples by optical fluorescence detection of differentially labelled proteins that are electrophoretically separated on the same gel. DIGE is an alternative to quantitation by MS-based methodologies and can circumvent their analytical limitations in areas such as intact protein analysis, (linear) detection over a wide range of protein abundances and, theoretically, applications where extreme sensitivity is needed. Thus, in quantitative proteomics DIGE is usually complementary to MS-based quantitation and has some distinct advantages. This review describes the basics of DIGE and its unique properties and compares it to MS-based methods in quantitative protein expression analysis. PMID:19003860

Timms, John F; Cramer, Rainer



Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Protein Banding Patterns among Rhizobium leguminosarum biovar phaseoli Strains Isolated from the Mexican Bean Phaseolus coccineus  

PubMed Central

Several rhizobial strains were isolated from Phaseolus coccineus root nodules and were determined to be Rhizobium leguminosarum biovar phaseoli strains after reinfection of the same host plant. These strains were characterized by cultural procedures (growth on different carbon sources and intrinsic antibiotic resistance) and electrophoretic procedures (sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins). Our results showed that these rhizobia are very similar to each other, especially in their electrophoretic protein banding patterns, suggesting that they might belong to isolated populations. Images

Arredondo-Peter, R.; Escamilla, E.



Phorbol diester-induced phosphorylation of nuclear matrix proteins in HL60 promyelocytes. Possible role in differentiation studied by cationic detergent gel electrophoresis  

SciTech Connect

Immortal HL60 promyelocytes are induced to differentiate to mortal adherent cells by a variety of agents which activate protein kinase C, including 12-O-tetradecanoylphorbol 13-acetate (TPA). In order to investigate the mechanism of this effect, we incubated HL60 cells with (/sup 32/P)orthophosphate with or without TPA and extracted their proteins with the cationic detergent benzyldimethyl-n-hexadecylammonium chloride prior to electrophoresis in a discontinuous polyacrylamide gel system in the first dimension. In this system, proteins migrate toward the cathode as a function of their molecular weight, and they are separated from other radioactive components which can obscure the pattern of protein phosphorylation on sodium dodecyl sulfate (SDS) gels. SDS gel electrophoresis was used in the second dimension, resulting in the clear resolution of a large number of proteins. TPA caused many changes in the pattern of protein phosphorylation in intact cells. Two proteins which prominently increased their incorporation of /sup 32/P were investigated in particular, and they were both found to be retained in the nuclear matrix following successive extraction of cells with Triton, digestion with DNase and RNase, and extraction with 2 M NaCl. These proteins migrated with apparent molecular weights of 80,000 and 33,000 on SDS gels, and are designated NP80 and NP33, respectively. NP80 was half-maximally phosphorylated after 7 min exposure to TPA, and half-maximally phosphorylated by 10 nM TPA. NP80 co-migrated with a faint Coomassie Blue-stained protein, and NP33 co-migrated with a more prominent protein. Several proteins incorporated less /sup 32/P when the cells were exposed to TPA, including one which was extracted from nuclei with the core histones and which co-migrated with histone H2A.

Macfarlane, D.E.



Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes  

PubMed Central

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

Trinkle-Mulcahy, Laura; Boulon, Severine; Lam, Yun Wah; Urcia, Roby; Boisvert, Francois-Michel; Vandermoere, Franck; Morrice, Nick A.; Swift, Sam; Rothbauer, Ulrich; Leonhardt, Heinrich; Lamond, Angus



Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients.  


A modified procedure for the two-dimensional electrophoretic analysis of basic polypeptides is described. This method uses isoelectric focusing with carrier ampholytes in the first dimension, and sodium dodecyl sulfate-electrophoresis in the second dimension. Counteraction of the cathodic drift is achieved by glass tube treatment (silanization), electrolyte modification (use of weak bases and acids), protection of the catholyte from carbon dioxide, and the addition of glycerol to the gel mix. Better resolution and reproducibility are obtained than with nonequilibrium pH gradient electrophoresis, since quasi equilibrium focusing can be obtained. PMID:8026444

Rabilloud, T



Biliary proteins: assessment of quantitative techniques and comparison in gallstone and nongallstone subjects  

Microsoft Academic Search

Although protein is the third most abundant solid in bile and is important in cholesterol crystal formation, methods for quantitating the concentration of total protein in bile have not been systematically evaluated. To establish a reliable protein assay for bile, we evaluated three protein assays (Lowry's method and the fluorescamine and Coomassie blue methods), and employed amino acid analysis as

Kiyoshi Yamazaki; Stephen P. Powers; Nicholas F. LaRusso


Sensitive, label-free protein assay using 1-ethyl-3-methylimidazolium tetrafluoroborate-supported microchip electrophoresis with laser-induced fluorescence detection.  


Based on the dimer-monomer equilibrium movement of the fluorescent dye Pyronin Y (PY), a rapid, simple, highly sensitive, label-free method for protein detection was developed by microchip electrophoresis with LIF detection. PY formed a nonfluorescent dimer induced by the premicellar aggregation of an anionic surfactant, SDS, however, the fluorescence intensity of the system increased dramatically when proteins such as BSA, bovine hemoglobin, cytochrome c, and trypsin were added to the solution due to the transition of dimer to fluorescent monomer. Furthermore, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF4) instead of PBS was applied as running buffers in microchip electrophoresis. Due to the excellent properties of EMImBF4, not only nonspecific protein adsorption was more efficiently suppressed, but also approximately ten-fold higher fluorescence intensity enhancement was obtained than that using PBS. Under the optimal conditions, detection limits for BSA, bovine hemoglobin, cytochrome c, and trypsin were 1.00x10(-6), 2x10(-6), 7x10(-7), and 5x10(-7) mg/mL, respectively. Thus, without covalent modification of the protein, a protein assay method with high sensitivity was achieved on microchips. PMID:18393338

Xu, Yuanhong; Li, Jing; Wang, Erkang



Immuno?Quantitative Polymerase Chain Reaction for Detection and Quantitation of Prion Protein  

Microsoft Academic Search

Immuno?polymerase chain reaction (PCR) is an extremely sensitive detection method, combining the specificity of antibody detection and the sensitivity of PCR. We have developed an immuno?quantitative PCR (iqPCR), exploiting real?time PCR technology, in order to improve this immuno?detection method and make it quantitative. To illustrate the advantages of iqPCR, we have compared it with a conventional enzyme linked immuno sorbent

Stéphanie Gofflot; Benaïssa El Moualij; Danièle Zorzi; Laurence Melen; Stefan Roels; Dominique Quatpers; Jacques Grassi; Emmanuel Vanopdenbosch; Ernst Heinen; Willy Zorzi



Quantitative determination of human cytomegalovirus target sequences in peripheral blood leukocytes by nested polymerase chain reaction and temperature gradient gel electrophoresis  

Microsoft Academic Search

A competitive nested PCR-temperature gradient gel electrophoresis protocol (nPCR\\/TGGE) has been es- tablished for the quantification of human cytomegalo- virus (HCMV) target sequences. The measurement was achieved by co-amplification of a defined copy number of an internal standard (st) and separation of st and wild- type (wt) amplimers by temperature gradient gel electro- phoresis (TGGE). The number of HCMV target

P. Schafer; Riidiger W. Braun; K. Mohring; Karsten Henco; Jie Kang; Thomas Wendland; J. E. Kuhn



Two-Dimensional Gel Electrophoresis Analyses of pH-Dependent Protein Expression in Facultatively Alkaliphilic Bacillus pseudofirmus OF4 Lead to Characterization of an SLayer Protein with a Role in Alkaliphily  

Microsoft Academic Search

The large majority of proteins of alkaliphilic Bacillus pseudofirmus OF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of




A replaceable microreactor for on-line protein digestion in a two-dimensional capillary electrophoresis system with tandem mass spectrometry detection  

PubMed Central

We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and ?-casein as model proteins.

Li, Yihan; Wojcik, Roza; Dovichi, Norman J.



Generation of a miniaturized free-flow electrophoresis chip based on a multi-lamination technique--isoelectric focusing of proteins and a single-stranded DNA fragment.  


Free-flow electrophoresis techniques have been applied for separations in various areas of chemistry and biochemistry. Here we focus on the generation of a free-flow electrophoresis chip and direct monitoring of the separation of different molecules in the separation bed of the miniaturized chip. We demonstrate a fast and efficient way to generate a low-cost micro-free-flow electrophoresis (?FFE) chip with a filling capacity of 9.5 ?L based on a multi-lamination technique. Separating webs realized by two transfer-adhesive tapes avoid the problem of gas bubbles entering the separation area. The chip is characterized by isoelectric focusing markers (IEF markers). The functionality of the chip is demonstrated by free-flow isoelectric focusing (FFIEF) of the proteins BSA (bovine serum albumin) and avidin and a single-stranded DNA (ssDNA) fragment in the pH range 3 to 10. The separation voltage ranges between 167 V cm(-1) and 422 V cm(-1), depending on the application. PMID:21912834

Walowski, Britta; Hüttner, Wilhelm; Wackerbarth, Hainer



A Comparison of Protein Quantitation Assays for Biopharmaceutical Applications  

Microsoft Academic Search

Dye-based protein determination assays are widely used to estimate protein concentration, however various reports suggest\\u000a that the response is dependent on the composition and sequence of the protein, limiting confidence in the resulting concentration\\u000a estimates. In this study a diverse set of model proteins representing various sizes of protein and covalent modifications,\\u000a some typical of biopharmaceuticals have been used to

J. E. Noble; A. E. Knight; A. J. Reason; A. Di Matola; M. J. A. Bailey



A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles  

NASA Astrophysics Data System (ADS)

It is now known that nanoparticles, when exposed to biological fluid, become coated with proteins and other biomolecules to form a `protein corona'. Recent systematic studies have identified various proteins that can make up this corona, but these nanoparticle-protein interactions are still poorly understood, and quantitative studies to characterize them are few in number. Here, we have quantitatively analysed the adsorption of human serum albumin onto small (10-20 nm in diameter) polymer-coated FePt and CdSe/ZnS nanoparticles by using fluorescence correlation spectroscopy. The protein corona forms a monolayer with a thickness of 3.3 nm. Proteins bind to the negatively charged nanoparticles with micromolar affinity, and time-resolved fluorescence quenching experiments show that they reside on the particle for ~100 s. These new findings deepen our quantitative understanding of the protein corona, which is of utmost importance in the safe application of nanoscale objects in living organisms.

Röcker, Carlheinz; Pötzl, Matthias; Zhang, Feng; Parak, Wolfgang J.; Nienhaus, G. Ulrich



Selective Extraction and Quantitation of Polyoxyethylene Detergents and its Application in Protein Determination  

Microsoft Academic Search

A simple, rapid and selective method for the nearly quantitative extraction of high amounts (10% w\\/v) of polyoxyethylene nonionic detergents like Triton X-100 is presented based on solvent extraction with 1, 2-dichloroethane. Protein determination after extraction of protein containing solutions can be accomplished using the Bradford assay or other standard methods. High protein recoveries can be obtained. The detergent analysis

Georg C. Terstappen; Maria-Regina Kula



VADAR: a web server for quantitative evaluation of protein structure quality  

Microsoft Academic Search

VADAR (Volume Area Dihedral Angle Reporter) is a comprehensive web server for quantitative protein structure evaluation. It accepts Protein Data Bank (PDB) formatted files or PDB accession numbers as input and calculates, identifies, graphs, reports and\\/ or evaluates a large number (>30) of key structural parameters both for individual residues and for the entire protein. These include excluded volume, accessible

Leigh Willard; Anuj Ranjan; Haiyan Zhang; Hassan Monzavi; Robert F. Boyko; Brian D. Sykes; David S. Wishart



Structure Modification and Functionality of Whey Proteins: Quantitative StructureActivity Relationship Approach  

Microsoft Academic Search

According to the original idea of quantitative structure-activity relation- ship, electric, hydrophobic, and structural parameters should be taken into con- sideration for elucidating functionality. Changes in these parameters are reflected in the property of protein solubility upon modification of whey proteins by heating. Although solubility is itself a functional property, it has been utilized to explain other functionalities of proteins.

S. Nakai; E. Li-chan



Urine sample preparation and protein profiling by two-dimensional electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectroscopy.  


Urine represents the most easily attainable and consequently one of the most common samples in clinical analysis and diagnostics. However, urine is also considered one of the most difficult proteomic samples to work with due to its highly variable contents, as well as the presence of various proteins in low abundance or modified forms. In this chapter, we describe simple protocols and troubleshooting tips for urinary protein preparation and profiling by two-dimensional electrophoresis or directly via matrix-assisted laser desorption ionization time of flight mass spectroscopy. Direct dilution, protein precipitation, ultrafiltration, and solid phase extraction in combination to the above profiling technologies serve the means for reliable proteomics analysis of one of the most significant yet very complex biological samples. PMID:18287772

Zerefos, Panagiotis G; Vlahou, Antonia



Two-dimensional gel electrophoresis analysis of mycelial cells treated with Tween 80: differentially expressed protein related to enhanced metabolite production.  


Two-dimensional gel electrophoresis identified 40 differentially expressed proteins which explained the mechanisms underlying the stimulatory effect of Tween 80 for exopolysaccharide production in the mycelium of an edible mushroom Pleurotus tuber-regium. The up-regulation of fatty acid synthase alpha subunit FasA might promote the synthesis of long-chain fatty acids and their incorporation into the mycelial cell membranes, increasing the membrane permeability. A down-regulation of Phospholipase D1 and an up-regulation of Hypothetical protein PGUG_02954 might mediate signal transduction between the mycelial cells and the extracellular stimulus (Tween 80). The down-regulated ATP-binding cassette transporter protein might function as pumps to extrude exopolysaccharide out of the cells that lead to a significant increase in its production. The present results explained how stimulatory agents like Tween 80 can increase mycelial cell membrane permeability to enhance the production of useful extracellular metabolites by submerged fermentation. PMID:23013510

Zhang, Bo-Bo; Chen, Lei; Cheung, Peter C K



Evaluation of two-dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples  

SciTech Connect

Laser microdissection (LM) allows the collection of homogeneous tissue- and cell specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there is as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryosectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.

Schad, Martina; Lipton, Mary S.; Giavalisco, Patrick; Smith, Richard D.; Kehr, Julia



On-line combination of capillary isoelectric focusing and capillary non-gel sieving electrophoresis using a hollow-fiber membrane interface: a novel two-dimensional separation system for proteins  

Microsoft Academic Search

A novel two-dimensional (2D) separation system for proteins was reported. In the system, a piece of dialysis hollow-fiber membrane was employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) and capillary non-gel sieving electrophoresis (CNGSE). The system is similar equivalent to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), by transferring the principal of 2D PAGE separation to the

Hechun Liu; Chun Yang; Qing Yang; Weibing Zhang; Yukui Zhang



Plasma Biomarker Discovery Using 3D Protein Profiling Coupled with Label-Free Quantitation  

PubMed Central

In-depth quantitative profiling of human plasma samples for biomarker discovery remains quite challenging. One promising alternative to chemical derivatization with stable isotope labels for quantitative comparisons is direct, label-free, quantitative comparison of raw LC–MS data. But, in order to achieve high-sensitivity detection of low-abundance proteins, plasma proteins must be extensively pre-fractionated, and results from LC–MS runs of all fractions must be integrated efficiently in order to avoid misidentification of variations in fractionation from sample to sample as “apparent” biomarkers. This protocol describes a powerful 3D protein profiling method for comprehensive analysis of human serum or plasma proteomes, which combines abundant protein depletion and high-sensitivity GeLC–MS/MS with label-free quantitation of candidate biomarkers.

Beer, Lynn A.; Tang, Hsin-Yao; Barnhart, Kurt T.; Speicher, David W.



iTRAQ-based quantitative analysis of protein mixtures with large fold change and dynamic range.  


Quantitation of changes in protein abundance is key to understanding the alterations that biological systems undergo and to discovering novel biomarkers. Currently, HPLC-MS/MS can be used to quantify changes in protein expression levels [Ong, S. E. and Mann, M., Nat. Chem. Biol. 2005, 1, 252-262]. Nevertheless, quantitative analysis of protein mixtures by HPLC-MS/MS is still hampered by the wide range of protein expression levels, the high dynamic range of protein concentrations and the lack of reliable quantitation algorithms [D'Ascenzo, M., et al. Brief. Funct. Genomic. Proteomic. 2008, 7, 127-135; Lin, W. T., et al., J. Proteome Res. 2006, 5, 2328-2338; Matthiesen, R., et al. J. Proteome Res. 2005, 4, 2338-2347; Yu, C. Y., et al. Nucleic Acids Res. 2007, 35, W707-W712]. In this context, we describe two different samples (4-protmix and 8-protmix) suitable for relative protein quantitation using iTRAQ. Using the 4-protmix, relative protein changes of up to 24-fold were measured. The 8-protmix allowed the quantitation of the relative protein changes in a mixture of proteins within the range of two orders of magnitude in concentration and ten-fold differences in relative abundance. We propose that the two samples are suited to test the iTRAQ quantitative proteomic workflow. We analyzed the iTRAQ samples with a LTQ Orbitrap using "higher energy collision-induced dissociation" fragmentation [Olsen, J. V., et al., Nat. Methods 2007, 4, 709-712] and quantified with Proteome Discoverer v.1.1 (Thermo Fisher Scientific). We believe that the presented protein mixtures will be useful to assess the performance of the iTRAQ-based quantitation proteomic strategy in any laboratory. PMID:20029838

Casado-Vela, Juan; Martínez-Esteso, María José; Rodriguez, Eva; Borrás, Eva; Elortza, Felix; Bru-Martínez, Roque



A quantitative, high-throughput screen for protein stability  

NASA Astrophysics Data System (ADS)

In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturationof purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.

Ghaemmaghami, S.; Fitzgerald, M. C.; Oas, T. G.



Quantitation of hepatic fatty acid-binding proteins by post-chromatographic ligand binding assay  

Microsoft Academic Search

A new procedure for the detection and quantitation of small molecular weight cytosolic fatty acid-binding proteins (FABP) in chromatographic fractions is described. Aliquots of the fractionated cytosol are incubated with radiolabeled pal- mitate and the unbound fatty acid ligand is quickly removed by addition of a dextran-gelatin-coated charcoal suspension. Quantitation of the FABP is accomplished by counting the protein-bound radioactivity

F. D. Morrow; R. J. Martin


Multiplexed Protein Quantitation in Saccharomyces cerevisiae Using Amine-reactive Isobaric Tagging Reagents  

Microsoft Academic Search

We describe here a multiplexed protein quantitation strat- egy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this method- ology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS\\/MS signature ions that support quantitation. In this study, we have

Philip L. Ross; Yulin N. Huang; Jason N. Marchese; Brian Williamson; Kenneth Parker; Stephen Hattan; Nikita Khainovski; Sasi Pillai; Subhakar Dey; Scott Daniels; Subhasish Purkayastha; Peter Juhasz; Stephen Martin; Michael Bartlet-Jones; Feng He; Allan Jacobson; Darryl J. Pappin



Interferences of suspended clay fraction in protein quantitation by several determination methods  

Microsoft Academic Search

Seven current methods of protein quantitation, Bradford (standard, micro, and 590\\/450nm ratio), Lowry, bicinchoninic acid (BCA), UV spectrophotometry at 280nm, and Quant-iT fluorescence-based determination, were compared with regard to their susceptibility to interferences due to the presence of suspended and not easily detectable clay particles. Bovine serum albumin (BSA) and Na-Wyoming montmorillonite were selected as model protein and reference clay,

I. Lozzi; A. Pucci; O. L. Pantani; L. P. D’Acqui; L. Calamai



Multi-Q:  A Fully Automated Tool for Multiplexed Protein Quantitation  

Microsoft Academic Search

Abstract The iTRAQ labeling method,combined with shotgun proteomic techniques represents a new,dimension inmultiplexed,quantitation for relative protein expression measurement ,in different cell states. To expedite the analysis of vast amounts of spectral data, we present a fully automated software package, called Multi-Q, for multiplexed iTRAQ-based quantitation in protein profiling. Multi-Q is designed as a generic platform that can accommodate ,various input

Wen-Ting Lin; Wei-Neng Hung; Yi-Hwa Yian; Kun-Pin Wu; Chia-Li Han; Yet-Ran Chen; Yu-Ju Chen; Ting-Yi Sung; Wen-Lian Hsu



Quantitative and qualitative differences in protein expression between papillary thyroid carcinoma and normal thyroid tissue.  


In order to better understand basic mechanisms of tumor development and identify potential new biomarkers, we have performed difference gel electrophoresis (DIGE) and peptide mass fingerprinting on pooled protein extracts from patients with papillary thyroid carcinoma (PTC) compared with matched normal thyroid tissue. Image analysis of DIGE gels comparing PTC and matched normal thyroid tissue protein indicated that 25% of the protein spots were differentially expressed at a 2.5-fold cutoff and 35% at two-fold. Comparison between two different pools of protein from normal thyroid tissues revealed differential protein expression of only 4% at 2.5-fold and 6% at two-fold cutoff. One hundred ninety-two protein spots were identified by MALDI-TOFMS, representing 90 distinct proteins. Excluding albumin, globins and thyroglobulin, imaging software determined 31 proteins to be differentially expressed at the two-fold (or greater) level. Individual gel comparisons (PTC vs. matched normal) from five patients established that 15/31 (48%) of these proteins exhibited statistically significant differential expression. Previously identified molecular markers in this group of proteins include cathepsin B, cytokeratin 19, and galectin-3. Novel differentially expressed proteins include S100A6, moesin, HSP70 (BiP), peroxiredoxin 2, protein phosphatase 2, selenium binding protein 1, vitamin D binding protein, and proteins involved in mitochondrial function. The use of two-dimensional gel electrophoresis (2DGE) revealed a significantly altered protein mass and/or pI in 10%-15% of proteins, suggesting alternatively spliced forms and other posttranslational modification of proteins revealed by this approach. We confirmed S100A6 as a potentially useful biomarker using immunohistochemical analysis (85% sensitivity and 69% specificity for distinguishing benign from malignant thyroid neoplasms). In summary, proteomic analysis of PTC using DIGE and mass spectrometry has confirmed several known biomarkers, uncovered novel potential biomarkers, and provided insights into global pathophysiologic changes in PTC. Many of the differences observed would not have been detected by genomic or other proteomic approaches. PMID:16788983

Brown, Lewis M; Helmke, Steve M; Hunsucker, Stephen W; Netea-Maier, Romana T; Chiang, Simon A; Heinz, David E; Shroyer, Kenneth R; Duncan, Mark W; Haugen, Bryan R



Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences.  


Separation of proteins by two-dimensional gel electrophoresis (2-DE) coupled with identification of proteins through peptide mass fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is the widely used technique for proteomic analysis. This approach relies, however, on the presence of the proteins studied in public-accessible protein databases or the availability of annotated genome sequences of an organism. In this work, we investigated the reliability of using raw genome sequences for identifying proteins by PMF without the need of additional information such as amino acid sequences. The method is demonstrated for proteomic analysis of Klebsiella pneumoniae grown anaerobically on glycerol. For 197 spots excised from 2-DE gels and submitted for mass spectrometric analysis 164 spots were clearly identified as 122 individual proteins. 95% of the 164 spots can be successfully identified merely by using peptide mass fingerprints and a strain-specific protein database (ProtKpn) constructed from the raw genome sequences of K. pneumoniae. Cross-species protein searching in the public databases mainly resulted in the identification of 57% of the 66 high expressed protein spots in comparison to 97% by using the ProtKpn database. 10 dha regulon related proteins that are essential for the initial enzymatic steps of anaerobic glycerol metabolism were successfully identified using the ProtKpn database, whereas none of them could be identified by cross-species searching. In conclusion, the use of strain-specific protein database constructed from raw genome sequences makes it possible to reliably identify most of the proteins from 2-DE analysis simply through peptide mass fingerprinting. PMID:14653859

Wang, Wei; Sun, Jibin; Nimtz, Manfred; Deckwer, Wolf-Dieter; Zeng, An-Ping



Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size  

SciTech Connect

In biofluids (e.g., blood plasma) nanoparticles are readily embedded in layers of proteins that can affect their biological activity and biocompatibility. Herein, we report a study on the interactions between human plasma proteins and nanoparticles with a controlled systematic variation of properties using stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS) based quantitative proteomics. Novel protocol has been developed to simplify the isolation of nanoparticle bound proteins and improve the reproducibility. Plasma proteins associated with polystyrene nanoparticles with three different surface chemistries and two sizes as well as for four different exposure times (for a total of 24 different samples) were identified and quantified by LC-MS analysis. Quantitative comparison of relative protein abundances were achieved by spiking an 18 O-labeled 'universal reference' into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantitation across the sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive pattern that classifies the nanoparticles based on their surface properties and size. In addition, data on the temporal study indicated that the stable protein 'corona' that was isolated for the quantitative analysis appeared to be formed in less than 5 minutes. The comprehensive results obtained herein using quantitative proteomics have potential implications towards predicting nanoparticle biocompatibility.

Zhang, Haizhen; Burnum, Kristin E.; Luna, Maria L.; Petritis, Brianne O.; Kim, Jong Seo; Qian, Weijun; Moore, Ronald J.; Heredia-Langner, Alejandro; Webb-Robertson, Bobbie-Jo M.; Thrall, Brian D.; Camp, David G.; Smith, Richard D.; Pounds, Joel G.; Liu, Tao



Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS).  


Identification of proteins separated by two-dimensional electrophoresis (2-DE) is a necessary task to overcome the purely descriptive character of 2-DE and a prerequisite to the construction of 2-DE databases in proteome projects. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver-stained protein spots by peptide mass fingerprinting. Unfortunately, proteins are modified by the silver staining procedure, leading to low sequence coverage. Omission of glutaraldehyde increased the sequence coverage, but this improved sequence coverage is still clearly below the sequence coverage starting with Coomassie Brilliant Blue (CBB) R-250-stained spots. Other factors additionally seem to modify proteins during silver staining. By decreasing the protein amount, the advantage of very sensitive detection on the gel is lost during identification, because the resulting low sequence coverage is not sufficient for secure identification. Low-quantity proteins can be identified better starting with CBB G-250 or Zn-imidazol-stained proteins. In contrast, for high-quantity CBB R-250-stained spots, a sequence coverage of up to 90% can be obtained by using only one cleaving enzyme, and up to 80% was reached for medium-quantity spots after combination of tryptic digest with Asp-N- and Glu-C digest. PMID:9638938

Scheler, C; Lamer, S; Pan, Z; Li, X P; Salnikow, J; Jungblut, P



Detection of tyrosine?phosphorylated proteins in hepatocellular carcinoma tissues using a combination of GST?Nck1?SH2 pull?down and two?dimensional electrophoresis.  


Tyrosine-phosphorylated proteins govern a host of cell functions, such as growth, division, adhesion and motility. We previously identified a group of Nck Src homology 2 (SH2) domain?binding proteins by combining the GST-Nck1-SH2 pull-down method with two-dimensional electrophoresis (2?DE) in hepatocellular carcinoma (HCC) tissues. In the present study, different methods and conditions for key procedures of GST-Nck1-SH2 pull-down and 2?DE were investigated and optimized. High-resolution results were obtained using the following conditions: a total amount of 100 µl GST-Nck1-SH2 fusion proteins/10 mg liver proteins to execute the pull-down procedure; 7 M urea and 2 M thiourea as lysis buffer; ultrafiltration depletion of interferential materials. Moreover, we performed a negative control experiment using GST-4T3 during the pull-down procedure, and further demonstrated that the proteins obtained using the aforementioned method interacted with Nck in a tyrosine phosphorylation-dependent manner. The optimized method offers a rapid, efficient alternative for the high?quantity screening of tyrosine-phosphorylated protein expression and solubility, which in turn facilitates future studies on tyrosine-phosphorylated proteins. PMID:23426619

Qiu, Fanghua; Huang, Dehong; Xiao, Hongguang; Qiu, Fangying; Lu, Liming; Nie, Jing



Compartmentalization and Quantitation of Protein in Human Milk1  

Microsoft Academic Search

Humanmilkproteinwas determinedbythree colorimetrie methods and by Kjeldahl analysis. The distri bution of nitrogen (N) and protein was determined within various milk compartments. Total N, whey, casein, non- protein nitrogen (NPN), cell N and N in the fat fraction were analyzed by micro-Kjeldahl analysis after a series of cen- trifugation and ultracentrifugation separations. Fresh milk samples (colostrum, transitional milk and mature



Protein interaction affinity determination by quantitative FRET technology.  


The dissociation constant, K(d) , is an important parameter for characterizing protein-protein interaction affinities. SUMOylation is one of the important protein post-translational modifications and it involves a multi-step enzymatic cascade reaction, resulting in peptide activation and substrate conjugation. Multiple covalent and non-covalent protein-protein interactions are involved in this cascade. Techniques involving Förster resonance energy transfer (FRET) have been widely used in biological studies in vitro and in vivo, and they are very powerful tools for elucidating protein interactions in many regulatory cascades. In our previous studies, we reported the attempt to develop a new method for the determination of the K(d) by FRET assay using the interaction of SUMO1 and its E2 ligase, Ubc9 as a test system. However, the generality and specifications of this new method have not been fully determined. Here we report a systematic approach for determining the dissociation constant (K(d) ) in the SUMOylation cascade and for further sensitivity and accuracy testing by the FRET technology. From a FRET donor to acceptor concentration ratio range of 4-40, the K(d) s of SUMO1 and Ubc9 consistently agree well with values from surface plasmon resonance and isothermal titration calorimetry. These results demonstrate the high sensitivity and accuracy of the FRET-based K(d) determination approach. This technology, therefore, can be used in general for protein-protein interaction dissociation constant determination. PMID:22711490

Song, Yang; Rodgers, V G J; Schultz, Jerome S; Liao, Jiayu



Functional Maps of Protein Complexes from Quantitative Genetic Interaction Data  

Microsoft Academic Search

Recently, a number of advanced screening technologies have allowed for the comprehensive quantification of aggravating and alleviating genetic interactions among gene pairs. In parallel, TAP-MS studies (tandem affinity purification followed by mass spectroscopy) have been successful at identifying physical protein interactions that can indicate proteins participating in the same molecular complex. Here, we propose a method for the joint learning

Sourav Bandyopadhyay; Ryan Kelley; Nevan J. Krogan; Trey Ideker



Neurodegenerative diseases: Quantitative predictions of protein-RNA interactions  

PubMed Central

Increasing evidence indicates that RNA plays an active role in a number of neurodegenerative diseases. We recently introduced a theoretical framework, catRAPID, to predict the binding ability of protein and RNA molecules. Here, we use catRAPID to investigate ribonucleoprotein interactions linked to inherited intellectual disability, amyotrophic lateral sclerosis, Creutzfeuld-Jakob, Alzheimer’s, and Parkinson’s diseases. We specifically focus on (1) RNA interactions with fragile X mental retardation protein FMRP; (2) protein sequestration caused by CGG repeats; (3) noncoding transcripts regulated by TAR DNA-binding protein 43 TDP-43; (4) autogenous regulation of TDP-43 and FMRP; (5) iron-mediated expression of amyloid precursor protein APP and ?-synuclein; (6) interactions between prions and RNA aptamers. Our results are in striking agreement with experimental evidence and provide new insights in processes associated with neuronal function and misfunction.

Cirillo, Davide; Agostini, Federico; Klus, Petr; Marchese, Domenica; Rodriguez, Silvia; Bolognesi, Benedetta; Tartaglia, Gian Gaetano



Site-specific quantitative analysis of cardiac mitochondrial protein phosphorylation.  


We report the development of a multiple-reaction monitoring (MRM) strategy specifically tailored to the detection and quantification of mitochondrial protein phosphorylation. We recently derived 68 MRM transitions specific to protein modifications in the respiratory chain, voltage-dependent anion channel, and adenine nucleotide translocase. Here, we have now expanded the total number of MRM transitions to 176 to cover proteins from the tricarboxylic acid cycle, pyruvate dehydrogenase complex, and branched-chain alpha-keto acid dehydrogenase complex. We utilized the transition set to analyze endogenous protein phosphorylation in human heart, mouse heart, and mouse liver. The data demonstrate the potential utility of the MRM workflow for studying the functional details of mitochondrial phosphorylation signaling. This article is part of a Special Issue entitled: From protein structures to clinical applications. PMID:23022582

Lam, Maggie P Y; Lau, Edward; Scruggs, Sarah B; Wang, Ding; Kim, Tae-Young; Liem, David A; Zhang, Jun; Ryan, Christopher M; Faull, Kym F; Ping, Peipei



Proteome analysis of human stomach tissue: separation of soluble proteins by two-dimensional polyacrylamide gel electrophoresis and identification by mass spectrometry.  


Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed in human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected by silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained with colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsin, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and is available at The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues. PMID:12210210

Ha, Geun Hyoung; Lee, Seung Uook; Kang, Deok Gyeong; Ha, Na-Young; Kim, Soon Hee; Kim, Jina; Bae, Jong Min; Kim, Jae Won; Lee, Chang-Won



[Analysis of insecticidal crystal proteins from Bacillus thuringiensis strain 4.0718 through two-dimensional gel electrophoresis and MALDI-TOF-mass spectrometry].  


The present study analyses the insecticidal crystal proteins (ICPs) from Bacillus thuringiensis strain 4.0718 through two-dimensional electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). By comparing and optimizing the composition of lysis solution, the volume of sample loading and the protocol for isoelectric focusing, a well-focused 2-DE map with high resolution and reproducibility was achieved for the first time. And after an tryptic enzymolisis and a test of part of protein spots by means of MALDI-TOF-MS, the peptides mass fingerprint (PMF) was obtained and, by referring to Swiss-Prot, Cry1Ac and Cry2Aa contained in Bt 4.0718 were identified, with their molecular weights 134160 Da and 71097 Da respectively. PMID:15989249

Song, Sheng; Xia, Li-qiu; Huang, Jiang-li; Sun, Yun-jun; Ding, Xue-zhi



Quantitative Assessment of In-solution Digestion Efficiency Identifies Optimal Protocols for Unbiased Protein Analysis.  


The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods. PMID:23792921

León, Ileana R; Schwämmle, Veit; Jensen, Ole N; Sprenger, Richard R



Evaluation of quail egg white riboflavin binding protein as a chiral selector in capillary electrophoresis by applying a modified partial filling technique.  


A preliminary evaluation of the enantioselective properties of quail egg yolk riboflavin binding protein (qRfBP) was carried out in capillary electrophoresis by using the complete filling technique. The most promising results obtained by this screening of nineteen chiral drugs were singled out with the aim of optimizing enantiomer separations by applying the partial filling technique, which allows operating at much higher protein concentrations without detection problems. The building of the separation zone in the partial filling technique has been modified in order to enable on-line monitoring, before each run, of the actual protein plug application velocity and, consequently, the building of a plug of the desired length. The electrophoretic conditions chosen gave opposite migration directions for the chiral selector and the analytes, with qRfBP migrating away from the detector. A polyvinyl alcohol-coated capillary was first totally filled with protein and the optimal plug length was obtained by further applying negative pressure together with positive voltage for the time needed. Separations of basic drugs were optimized by using protein concentrations ranging from 200 microM up to 900 microM and different plug lengths, while the running buffer pH (6.0), temperature (25 degrees C) and operating voltage (+20 kV) were kept constant. The enantioresolution of all solutes was affected by both the chiral selector concentration and protein plug length. Baseline separations were obtained for oxprenolol, prilocaine and bupivacaine. PMID:10532342

De Lorenzi, E; Massolini, G; Quaglia, M; Galbusera, C; Caccialanza, G



Quantitative proteomic analysis of cold-responsive proteins in rice.  


Rice is susceptible to cold stress and with a future of climatic instability we will be unable to produce enough rice to satisfy increasing demand. A thorough understanding of the molecular responses to thermal stress is imperative for engineering cultivars, which have greater resistance to low temperature stress. In this study we investigated the proteomic response of rice seedlings to 48, 72 and 96?h of cold stress at 12-14°C. The use of both label-free and iTRAQ approaches in the analysis of global protein expression enabled us to assess the complementarity of the two techniques for use in plant proteomics. The approaches yielded a similar biological response to cold stress despite a disparity in proteins identified. The label-free approach identified 236 cold-responsive proteins compared to 85 in iTRAQ results, with only 24 proteins in common. Functional analysis revealed differential expression of proteins involved in transport, photosynthesis, generation of precursor metabolites and energy; and, more specifically, histones and vitamin B biosynthetic proteins were observed to be affected by cold stress. PMID:21433000

Neilson, Karlie A; Mariani, Michael; Haynes, Paul A



Protein adsorption dynamics in cation-exchange chromatography quantitatively studied by confocal laser scanning microscopy  

Microsoft Academic Search

A self-contained research system based on the technique of confocal laser scanning microscopy (CLSM) was put up to quantitatively analyze the dynamics of protein adsorption to porous cation exchanger by mathematical modeling. Bovine serum albumin adsorption to the cation exchanger SP Sepharose FF was performed by batch adsorption and micro-flow cell in which protein concentration in single absorbent was visualized

Kun Yang; Shu Bai; Yan Sun



Quantitative trait loci influencing protein and starch concentration in the Illinois Long Term Selection maize strains  

Microsoft Academic Search

A study was initiated to determine the number, chromosomal location, and magnitude of effect of QTL (quantitative trait loci or locus depending on context) controlling protein and starch concentration in the maize (Zea mays L.) kernel. Restriction fragment length polymorphism (RFLP) analysis was performed on 100 F3 families derived from a cross of two strains, Illinois High Protein (IHP), X

I. L. Goldman; T. R. Rocheford; J. W. Dudley



The Association of Biomolecular Resource Facilities Proteomics Research Group 2006 Study: Relative Protein Quantitation  

Microsoft Academic Search

The determination of differences in relative protein abun- dance is a critical aspect of proteomics research that is increasingly used to answer diverse biological questions. The Association of Biomolecular Resource Facilities Pro- teomics Research Group 2006 study was a quantitative proteomics project in which the aim was to determine the identity and the relative amounts of eight proteins in two

Christoph W. Turck; Arnold M. Falick; Jeffrey A. Kowalak; Kathryn S. Lilley; Brett S. Phinney; Susan T. Weintraub; H. Ewa Witkowska; N. A. Yates




EPA Science Inventory

An immunoassay method is described for the quantitative determination of synapsin I (protein I) and of a 36,000-dalton membrane protein from rat brain synaptic vesicles. The samples are spotted on nitrocellulose membrane filters, incubated sequentially with specific antibodies an...


Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions  

Microsoft Academic Search

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through

Lance M Hellman; Michael G Fried



Gel Electrophoresis on a Budget to Dye For  

NSDL National Science Digital Library

Gel electrophoresis is one of the most important tools used in molecular biology and has facilitated the entire field of genetic engineering by enabling the separation of nucleic acids and proteins. However, commercial electrophoresis kits can cost up to

Yu, Julie H.



An evaluation of protein assays for quantitative determination of drugs  

Microsoft Academic Search

We have evaluated the response of six protein assays [the biuret, Lowry, bicinchoninic acid (BCA), Coomassie Brilliant Blue (CBB), Pyrogallol Red–Molybdate (PRM), and benzethonium chloride (BEC)] to 21 pharmaceutical drugs. The drugs evaluated were analgesics (acetaminophen, aspirin, codeine, methadone, morphine and pethidine), antibiotics (amoxicillin, ampicillin, gentamicin, neomycin, penicillin G and vancomycin), antipsychotics (chlorpromazine, fluphenazine, prochlorperazine, promazine and thioridazine) and water-soluble

Katherine M. Williams; Sarah J. Arthur; Gillian Burrell; Fionnuala Kelly; Darren W. Phillips; Thomas Marshall



A new densitometric procedure to measure protein levels in tissue slices used in quantitative autoradiography.  


A new quantitative staining technique for the assay of protein in tissue sections using the dye Coomassie brilliant blue G 250 is discussed. This densitometric procedure uses commercially available hardware and software employed in the quantitation of receptor autoradiographs. Rather than assuming a homogeneous distribution of protein in the tissue section, regional levels of protein are measured in the same tissue slice used to produce the autoradiograph. Additionally, the process of staining tissue with Coomassie blue for protein is reversible; the tissue can be destained after the measurement of protein is complete and then restained with a standard histological stain such as Cresyl violet. This technique is a more reliable method for normalizing receptor densities by tissue protein levels and allows for a more accurate comparison between QAR and membrane binding techniques. PMID:2838130

Miller, J A; Curella, P; Zahniser, N R



Quantitative capillary zone electrophoresis method for the precise determination of charge differences arising from the manufacture of heparan-N-sulfatase.  


A rapid and reproducible high-resolution capillary zone electrophoresis (CZE) method capable of resolving the charge isoforms of intact heparan-N-sulfatase (HNS) has been developed to monitor the charge consistency across different batches of HNS. Separation was carried out using a bare fused silica capillary with a buffer system composed of 25 mM Tris, pH 8.0. This CZE method allowed the separation and integration of 14 peaks, each arising from differences in the amount of sialic-acid and mannose-6-phosphate bearing glycoforms, which were confirmed using enzymatically modified samples. Standard conditioning and rinsing conditions of the capillary were used to achieve optimal repeatability. Excellent day-to-day precision was obtained for migration times of each peak relative to the electroosmotic flow marker with relative standard deviation (RSD)? 0.5%. The precision of the relative peak areas (peak area percentages) ranged from 0.6% to 2.8% RSD for the major isoforms (peaks 3-12), from 4.0% to 5.0% RSD for peaks 1 and 2, and from 7.4% to 23.2% RSD for peaks 13 and 14. The method was able to discriminate charge variation across different batches of HNS, including those with both significant and minor process changes. PMID:23917036

Roseman, Daniel S; Weinberger, Robert



Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system.  


Precise and accurate quantification of protein expression levels in a complex biological setting is challenging. Here, we describe a method for absolute quantitation of endogenous proteins in cell lysates using an automated capillary immunoassay system, the size-based Simple Western system (recently developed by ProteinSimple). The method was able to accurately measure the absolute amounts of target proteins at picogram or sub-picogram levels per nanogram of cell lysates. The measurements were independent of the cell matrix or the cell lysis buffer and were not affected by different antibody affinities for their specific epitopes. We then applied this method to quantitate absolute levels of expression of protein kinase C (PKC) isoforms in LNCaP and U937 cells, two cell lines used extensively for probing the downstream biological responses to PKC targeted ligands. Our absolute quantitation confirmed the predominance of PKC? in both cells, supporting the important functional role of this PKC isoform in these cell lines. The method described here provides an approach to accurately quantitate levels of protein expression and correlate protein level with function. In addition to enhanced accuracy relative to conventional Western analysis, it circumvents the distortions inherent in comparison with signal intensities from different antibodies with different affinities. PMID:23896461

Chen, Jin-Qiu; Heldman, Madeleine R; Herrmann, Michelle A; Kedei, Noemi; Woo, Wonhee; Blumberg, Peter M; Goldsmith, Paul K



Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes.  


The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments. PMID:18936248

Trinkle-Mulcahy, Laura; Boulon, Séverine; Lam, Yun Wah; Urcia, Roby; Boisvert, François-Michel; Vandermoere, Franck; Morrice, Nick A; Swift, Sam; Rothbauer, Ulrich; Leonhardt, Heinrich; Lamond, Angus



Quantitative trait loci for ?-conglycinin (7S) and glycinin (11S) fractions of soybean storage protein  

Microsoft Academic Search

Glycinin (11S) and ?-conglycinin (7S) are important seed storage proteins in soybean [Glycine max (L.) Merr.]. A major limitation of soybean seed storage proteins is their low levels of the sulfur-containing amino acids,\\u000a methionine and cysteine, which are important nutritional components of protein mea. Glycinin contains significantly more S-containing\\u000a amino acids than does ?-conglycinin. Thus, detection of quantitative trait loci

D. R. Panthee; P. Kwanyuen; C. E. Sams; D. R. West; A. M. Saxton; V. R. Pantalone



Capillary electrophoresis for meat species differentiation.  


A sodium dodecyl sulfate (SDS) polymer-filled capillary gel electrophoresis (CE-SDS) method was developed and optimized for the determination of meat proteins for species differentiation. Sarcoplasmic proteins were extracted with cold bidistilled deionized water and myofibrillar proteins with 0.6 M NaCl/0.01 M phosphate buffer with 0.5% polyphosphates at pH 6 from raw beef, turkey, and pork muscles. Samples were prepared for CE-SDS and the experimental conditions, including sample size, applied voltage, reducing agent, and its concentration, were obtained after a univariate optimization process. Separation of the sarcoplasmic and myofibrillar meat proteins was achieved with the optimized conditions of the CE-SDS method developed. The coefficient of variation was less than 1.15% in migration time for all peaks and less than 8.5% in area percentage. The CE-SDS sarcoplasmic protein profiles that resulted were specific for each species both qualitatively and quantitatively and could be employed for differentiation and identification purposes. This CE-SDS method can be used by regulatory agencies for rapid analysis of meat proteins to identify meat species. Automation, fast separation, and on-line data analysis are major advantages of this technique. PMID:9627836

Cota-Rivas, M; Vallejo-Cordoba, B V


Secondary Reactions and Strategies to Improve Quantitative Protein Footprinting  

SciTech Connect

Hydroxyl radical-mediated footprinting permits detailed examination of structure and dynamic processes of proteins and large biological assemblies, as changes in the rate of reaction of radicals with target peptides are governed by changes in the solvent accessibility of the side-chain probe residues. The precise and accurate determination of peptide reaction rates is essential to successfully probing protein structure using footprinting. In this study, we specifically examine the magnitude and mechanisms of secondary oxidation occurring after radiolytic exposure and prior to mass spectrometric analysis. Secondary oxidation results from hydrogen peroxide and other oxidative species generated during radiolysis, significantly impacting the oxidation of Met and Cys but not aromatic or other reactive residues. Secondary oxidation of Met with formation of sulfoxide degrades data reproducibility and inflates the perceived solvent accessibility of Met-containing peptides. It can be suppressed by adding trace amounts of catalase or millimolar Met-NH{sub 2} (or Met-OH) buffer immediately after irradiation; this leads to greatly improved adherence to first-order kinetics and more precise observed oxidation rates. The strategy is shown to suppress secondary oxidation in model peptides and improve data quality in examining the reactivity of peptides within the Arp2/3 protein complex. Cysteine is also subject to secondary oxidation generating disulfide as the principal product. The disulfides can be reduced before mass spectrometric analysis by reducing agents such as TCEP, while methionine sulfoxide is refractory to reduction by this reagent under typical reducing conditions.

Xu,G.; Kiselar, J.; He, Q.; Chance, M.



Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry.  

PubMed Central

A number of the culture filtrate proteins secreted by Mycobacterium tuberculosis are known to contribute to the immunology of tuberculosis and to possess enzymatic activities associated with pathogenicity. However, a complete analysis of the protein composition of this fraction has been lacking. By using two-dimensional polyacrylamide gel electrophoresis, detailed maps of the culture filtrate proteins of M. tuberculosis H37Rv were generated. In total, 205 protein spots were observed. The coupling of this electrophoretic technique with Western blot analysis allowed the identification and mapping of 32 proteins. Further molecular characterization of abundant proteins within this fraction was achieved by N-terminal amino acid sequencing and liquid chromatography-mass spectrometry. Eighteen proteins were subjected to N-group analysis; of these, only 10 could be sequenced by Edman degradation. Among the most interesting were a novel 52-kDa protein demonstrating significant homology to an alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708, a 25-kDa protein corresponding to open reading frame 28 of the M. tuberculosis cosmid MTCY1A11, and a 31-kDa protein exhibiting an amino acid sequence identical to that of antigen 85A and 85B. This latter product migrated with an isoelectric point between those of antigen 85A and 85C but did not react with the antibody specific for this complex, suggesting that there is a fourth member of the antigen 85 complex. Novel N-terminal amino acid sequences were obtained for three additional culture filtrate proteins; however, these did not yield significant homology to known protein sequences. A protein cluster of 85 to 88 kDa, recognized by the monoclonal antibodies IT-57 and IT-42 and known to react with sera from a large proportion of tuberculosis patients, was refractory to N-group analysis. Nevertheless, mass spectrometry of peptides obtained from one member of this complex identified it as the M. tuberculosis KatG catalase/peroxidase. Thus, the detailed mapping of M. tuberculosis proteins, combined with state-of-the-art analytical techniques such as mass spectrometry, provides a basis for further analysis and rapid identification of biologically relevant molecules.

Sonnenberg, M G; Belisle, J T



Advances in agarose gel electrophoresis of serum lipoproteins  

Microsoft Academic Search

Agarose gel electrophoresis has been extensively employed by researchers to gain a greater understanding of lipoprotein biology and its relationship to cardiovascular disease. Advances in this technique have been made in the visualization and quantitation of separated lipoproteins, in the use of agarose gel electrophoresis for detection and quantitation of apolipoproteins of the separated lipoproteins, and in the detection of

Phillip Greenspan; Fei-wen Mao; Beung-Ho Ryu; Robert L. Gutman



Getting the Most out of Electrophoresis Units  

ERIC Educational Resources Information Center

|At Oklahoma City Community College, they have developed gel electrophoresis activities that support active learning of many scientific concepts, including: pH, electrolysis, oxidation reduction, electrical currents, potentials, conductivity, molarity, gel electrophoresis, DNA and protein separation, and DNA fingerprinting. This article presents…

Mulvihill, Charlotte



The effect of weight loss on protein profiles of gastrocnemius muscle in rabbits: a study using 1D electrophoresis and peptide mass fingerprinting.  


The study of physiological changes occurring during selection contributes to an improved understanding of relationships leading to efficiencies in animal production. To investigate the effects of food restriction in gastrocnemius muscle protein expression, 20% weight reduction was induced in New Zealand White (meat producing) and wild rabbits, using one-dimensional gel electrophoresis and peptide mass fingerprinting. Lower expression levels of myosin heavy chains were found in the Wild Rabbits Restricted Group, while myosin light chain and alpha-crystallin proteins were not detected in restricted groups. Glyceraldeyde-3-phosphate dehydrogenase and glycogen phosphorylase expression levels were similar for all experimental groups. Phosphopyruvate hydratase beta was not detected in the wild rabbit restricted diet group. Pyruvate kinase levels were 50% lower in the New Zealand Restricted group. LIM protein detection was absent in the control New Zealand group. Results also show relevance of actin in preserving muscle structure in depressed food availability, the sensitivity of both myosin light chain and alpha-crystallin protein to restricted feed and the role of PK in the resistance of New Zealand rabbits to food restriction. PMID:19175456

Almeida, A M; van Harten, S; Campos, A; Coelho, A V; Cardoso, L A



Experimental design applied for the simultaneous analysis of whey proteins and caseins of binary and ternary milk mixtures by capillary electrophoresis.  


A capillary electrophoresis method for the simultaneous separation of caseins, whey proteins, and para-kappa-casein that appears during the manufacture of cheese was optimized using the response surface methodology. The parameters selected for this study were pH, voltage, and temperature. Under the optimized conditions (30 kV at 20 degrees C with 10 mM phosphate buffer at pH 3) casein proteins alpha(s)-casein; beta-casein, including genetic variants A1, A2, and B, kappa-CN, and para-kappa-CN; and whey proteins alpha-lactalbumin and beta-lactoglobulin (A and B variants) were separated. The method was applied successfully to skim milk, to casein precipitated at pH 4.6, and to a model sample containing rennet casein and milk. The milk used was of three types: cow, ewe, and goat. The present procedure can be easily applied to the separation of the major bovine, ovine, and caprine milk proteins in binary and ternary milk mixtures. PMID:16042123

Rodríguez-Nogalesa, José M; Revilla, Isabel; Vivar-Quintana, Ana M



Enzyme electrophoresis, sero- and subtyping, and outer membrane protein characterization of two Neisseria meningitidis strains involved in laboratory-acquired infections.  

PubMed Central

Two cases of laboratory-acquired infections due to Neisseria meningitidis were suspected to have occurred in two French hospitals. The first case occurred shortly, i.e., 3 days, after one strain had been handled by a laboratory technician, and the link between this strain and the strain causing meningitis was easily established. In the second case, infection occurred 3 weeks after 10 strains had been handled by a technician. In this case, it was necessary to use high-resolution markers in order to establish the link between the infecting strain and 1 of the 10 strains handled. The antigenic formulae of the two infecting strains (serogroup:serotype:subtype) were, respectively, C:NT:P1.12 and B:2a:P1.2. Outer membrane protein profile analysis and multilocus enzyme electrophoresis unequivocally confirmed the identity of the respective strains. Images

Guibourdenche, M; Darchis, J P; Boisivon, A; Collatz, E; Riou, J Y



Top-down quantitation and characterization of SILAC-labeled proteins  

Microsoft Academic Search

Stable isotope labeling by amino acids in cell culture (SILAC) has become a popular labeling strategy for peptide quantitation\\u000a in proteomics experiments. If the SILAC technology could be extended to intact proteins, it would enable direct quantitation\\u000a of their relative expression levels and of the degree of modification between different samples. Here we show through modeling\\u000a and experiments that SILAC

Leonie F. Waanders; Stefan Hanke; Matthias Mann



Quantitative determination of amino acids in protein hydrolyzates by thin-layer chromatography  

Microsoft Academic Search

Conclusions  A micro method for the quantitative determination of amino acids in protein hydrolyzates has been proposed which permits the\\u000a analysis of two hydrolyzates to be performed in 4–5 h. After the separation of the amino acids by TLC in cellulose, they are\\u000a determined quantitatively by the colorimetric method using a MKMF-1 microphotocolorimeter. Proline and tryptophan are determined\\u000a separately with the

M. D. Lutsik; I. I. Litvin; V. A. Monastyrskii; Ya. I. Aleksevich



Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions  

PubMed Central

Protein interactions are involved in all cellular processes. Their efficient and reliable characterization is therefore essential for understanding biological mechanisms. In this study, we show that combining bacterial artificial chromosome (BAC) TransgeneOmics with quantitative interaction proteomics, which we call quantitative BAC–green fluorescent protein interactomics (QUBIC), allows specific and highly sensitive detection of interactions using rapid, generic, and quantitative procedures with minimal material. We applied this approach to identify known and novel components of well-studied complexes such as the anaphase-promoting complex. Furthermore, we demonstrate second generation interaction proteomics by incorporating directed mutational transgene modification and drug perturbation into QUBIC. These methods identified domain/isoform-specific interactors of pericentrin- and phosphorylation-specific interactors of TACC3, which are necessary for its recruitment to mitotic spindles. The scalability, simplicity, cost effectiveness, and sensitivity of this method provide a basis for its general use in small-scale experiments and in mapping the human protein interactome.

Hubner, Nina C.; Bird, Alexander W.; Cox, Jurgen; Splettstoesser, Bianca; Bandilla, Peter; Poser, Ina



Detection of seminal fluid proteins in the bed bug, Cimex lectularius, using two-dimensional gel electrophoresis and mass spectrometry  

PubMed Central

SUMMARY The global increase of the human parasite, the common bed bug Cimex lectularius, calls for specific pest control target sites. The bed bug is also a model species for sexual conflict theory which suggests seminal fluids may be highly diverse. The species has a highly unusual sperm biology and seminal proteins may have unique functions. 1-D PAGE gels showed 40 to 50% band sharing between C. lectularius and another cimicid species, Afrocimex constrictus. However, adult, sexually rested C. lectularius males were found to store 5 to 7?g of seminal protein and with only 60?g of protein we obtained informative 2-D PAGE gels. These showed 79% shared protein spots between two laboratory populations, and more than half of the shared protein spots were detected in the mated female. Further analysis using liquid chromatography electrospray ionisation tandem mass spectrometry revealed that 26.5% of the proteins had matches among arthropods in data bases and 14.5% matched Drosophila proteins. These included ubiquitous proteins but also those more closely associated with reproduction such as moj 29, ubiquitin, the stress-related elongation factor EF-1alpha, a protein disulfide isomerase and an antioxidant, Peroxiredoxin 6.




Detection of seminal fluid proteins in the bed bug, Cimex lectularius, using two-dimensional gel electrophoresis and mass spectrometry.  


The global increase of the human parasite, the common bed bug Cimex lectularius, calls for specific pest control target sites. The bed bug is also a model species for sexual conflict theory which suggests that seminal fluids may be highly diverse. The species has a highly unusual sperm biology and seminal proteins may have unique functions. One-dimensional PAGE gels showed 40-50% band sharing between C. lectularius and another cimicid species, Afrocimex constrictus. However, adult, sexually rested C. lectularius males were found to store 5-7 microg of seminal protein and with only 60 microg of protein we obtained informative 2-D PAGE gels. These showed 79% shared protein spots between 2 laboratory populations, and more than half of the shared protein spots were detected in the mated female. Further analysis using liquid chromatography electrospray ionization tandem mass spectrometry revealed that 26.5% of the proteins had matches among arthropods in databases and 14.5% matched Drosophila proteins. These included ubiquitous proteins but also those more closely associated with reproduction such as moj 29, ubiquitin, the stress-related elongation factor EF-1 alpha, a protein disulfide isomerase and an antioxidant, Peroxiredoxin 6. PMID:19091156

Reinhardt, K; Wong, C H; Georgiou, A S



Quantitative Profiling and Identification of Plasma Proteins of Spinocerebellar Ataxia Type 2 Patients.  


Background: Spinocerebellar ataxia type 2 (SCA2) is an autosomal-dominant hereditary ataxia characterized by progressive gait and limb ataxia, dysarthria, slow saccades, neuropathy and dementia. The expansion of trinucleotide CAG repeats in the coding region of the ATXN-2 gene leads to expanded polyglutamine stretch in the mutated protein which causes neuronal death. Objective: In this study, we investigated the blood plasma of SCA2 patients to find protein biomarkers. Methods: Thirty-two ataxia patients clinically suspected for SCA2 were evaluated by the International Co-operative Ataxia Rating Scale followed by genetic analysis using PCR. Plasma proteomics of SCA2 patients and age- and gender-matched healthy controls was done using 2D-difference in-gel electrophoresis, LC-MS/MS and Western blot. Results: Genetic analysis confirmed 10 of 32 suspected SCA2 patients. Proteomic data revealed nine differentially expressed proteins in SCA2. These proteins find good association with oxidative stress, calcium-dependent apoptosis, neuropathy, and cognitive impairment in SCA2 patients. Interestingly, the elevated levels of the voltage-dependent calcium channel ?-3 subunit showed a direct correlation with calcium-generated apoptosis of Purkinje cells. The cognitive deficit, a common symptom in SCA2 patients, seems to correlate with decreased levels of transthyretin and retinol-binding protein-4. Conclusions: Some of these identified proteins in SCA2 can be useful for therapeutic, diagnostic and prognostic purposes. PMID:23735416

Swarup, Vishnu; Srivastava, Achal K; Padma, Madakasira V; Moganty, Rajeswari R



Labeling of capsid proteins and genomic RNA of human rhinovirus with two different fluorescent dyes for selective detection by capillary electrophoresis.  


During uncoating of human rhinoviruses, the innermost capsid protein VP4 and the genomic RNA are released from the viral protein shell. This process gives rise to subviral particles that are composed of the remaining three capsid proteins VP1, VP2, and VP3. The process is believed to take place in a sequential manner in that first VP4 is expelled resulting in A-particles sedimenting at 135S followed by the RNA resulting in B-particles sedimenting at 80S. Aiming at ultimately analyzing this process in vivo, we introduced two different fluorophores into the RNA and the viral capsid proteins, respectively. Incubation of the virus with RiboGreen resulted in formation of a RNA-dye complex with lambda(ex)/lambda(em) = 500/525 nm, whereas subsequent derivatization of the viral protein shell in the same sample with AMCA-S introduced a label with lambda(ex)/lambda(em) = 345-350/440-460 nm. In this way, both viral components could be selectively detected via fluorescence in a capillary electrophoresis system. The intact virus delivers two superimposed signals in the electropherogram. Derivatization of the free amino groups of the capsid proteins partially preserved the bioaffinity of the virus toward a synthetic receptor fragment, an artificial recombinant concatemer of repeat number 3 of the very low density lipoprotein receptor. Between 10 and 20% of the infectivity were recovered after labeling when compared to native virus. In addition to analysis of factors influencing the stability of the virus by CE, double-labeled virions might be useful for the investigation of the uncoating process by real-time confocal fluorescence microscopy. PMID:15595880

Kremser, Leopold; Petsch, Martina; Blaas, Dieter; Kenndler, Ernst



Analysis of arsenic stress-induced differentially expressed proteins in rice leaves by two-dimensional gel electrophoresis coupled with mass spectrometry.  


In the present study, we have investigated the protein expression profile of rice leaves under arsenic (As) stress. Two-week-old rice seedlings were exposed to two concentrations of arsenate (50 or 100 microM), and leaf samples were collected 4d after treatment. To elucidate the As stress-induced differentially expressed proteins in rice leaves, proteins were extracted from the control and treated samples, separated by two-dimensional gel electrophoresis (2-DE), and visualized by staining with Coomassie Brilliant Blue (CBB). A total of 14 protein spots showed reproducible changes in expression of at least 1.5-fold when compared to the control and showed a similar expression pattern in both treatments. Of these 14 spots, 8 were up-regulated and 6 were down-regulated following exposure to As. These proteins were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). The increased expression of several proteins associated with energy production and metabolism suggests that higher energy is required for activation of the metabolic processes in leaves exposed to As. On the other hand, results from the 2-DE analysis, combined with immunoblotting, clearly revealed that the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit was significantly decreased under As stress. Thus, the down-regulation of RuBisCO and chloroplast 29 kDa ribonucleoproteins might be the possible causes of the decreased photosynthesis rate under As stress. PMID:19948354

Ahsan, Nagib; Lee, Dong-Gi; Kim, Kyung-Hee; Alam, Iftekhar; Lee, Sang-Hoon; Lee, Ki-Won; Lee, Hyoshin; Lee, Byung-Hyun



Absolute quantitation of isoforms of post-translationally modified proteins in transgenic organism.  


Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (P(isf)) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent (14)N-coded synthetic peptide standards and (15)N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (T(isf)) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (R(aqu)). The P(isf) was finally determined by integrating the two empirically measured variables using the following equation: P(isf) = T(isf) · R(aqu). The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants. PMID:22442259

Li, Yaojun; Shu, Yiwei; Peng, Changchao; Zhu, Lin; Guo, Guangyu; Li, Ning



Quantitative Proteomic Analysis of Ovarian Cancer Cells Identified Mitochondrial Proteins Associated with Paclitaxel Resistance  

PubMed Central

Paclitaxel has been widely used as an anti-mitotic agent in chemotherapy for a variety of cancers and adds substantial efficacy as the first-line chemotherapeutic regimen for ovarian cancers. However, the frequent occurrence of paclitaxel resistance limits its function in long-term management. Despite abundant clinical and cellular demonstration of paclitaxel resistant tumors, the molecular mechanisms leading to paclitaxel resistance are poorly understood. Using genomic approaches, we have previously identified an association between a BTB/POZ gene, Nac1, and paclitaxel resistance in ovarian cancer. The experiments presented here have applied multiple quantitative proteomic methods to identify protein changes associated with paclitaxel resistance and Nac1 function. The SKOV-3 ovarian serous carcinoma cell line, which has inducible expression of dominant negative Nac1, was used to determine the paclitaxel treatment associated changes in the presence and absence of functional Nac1. Quantitative proteomic analyses were performed using iTRAQ labeling and mass spectrometry. Two label-free quantitative proteomic methods: LC-MS and spectral count were used to increase confidence of proteomic quantification. A total of 1371 proteins were quantified by at least one of the quantitative proteomic methods. Candidate proteins related to paclitaxel and NAC1 function were identified in this study. Go analysis of the protein changes identified upon paclitaxel resistance revealed that cell component enrichment related to mitochondria. Moreover, tubulin and mitochondrial proteins were the major cellular components with changes associated with paclitaxel treatment. This suggests that mitochondria may play a role in paclitaxel resistance.

Tian, Yuan; Tan, Aik-Choon; Sun, Xiaer; Olson, Matthew T; Xie, Zhi; Jinawath, Natini; Chan, Daniel W.; Shih, Ie-Ming; Zhang, Zhen; Zhang, Hui



A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.  


Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity. PMID:21520910

Bohler, Sacha; Sergeant, Kjell; Hoffmann, Lucien; Dizengremel, Pierre; Hausman, Jean-Francois; Renaut, Jenny; Jolivet, Yves



High Blood Pressure Effects on the Blood to Cerebrospinal Fluid Barrier and Cerebrospinal Fluid Protein Composition: A Two-Dimensional Electrophoresis Study in Spontaneously Hypertensive Rats  

PubMed Central

The aim of the present work is to analyze the cerebrospinal fluid proteomic profile, trying to find possible biomarkers of the effects of hypertension of the blood to CSF barrier disruption in the brain and their participation in the cholesterol and ?-amyloid metabolism and inflammatory processes. Cerebrospinal fluid (CSF) is a system linked to the brain and its composition can be altered not only by encephalic disorder, but also by systemic diseases such as arterial hypertension, which produces alterations in the choroid plexus and cerebrospinal fluid protein composition. 2D gel electrophoresis in cerebrospinal fluid extracted from the cistern magna before sacrifice of hypertensive and control rats was performed. The results showed different proteomic profiles between SHR and WKY, that ?-1-antitrypsin, apolipoprotein A1, albumin, immunoglobulin G, vitamin D binding protein, haptoglobin and ?-1-macroglobulin were found to be up-regulated in SHR, and apolipoprotein E, transthyretin, ?-2-HS-glycoprotein, transferrin, ?-1?-glycoprotein, kininogen and carbonic anhidrase II were down-regulated in SHR. The conclusion made here is that hypertension in SHR produces important variations in cerebrospinal fluid proteins that could be due to a choroid plexus dysfunction and this fact supports the close connection between hypertension and blood to cerebrospinal fluid barrier disruption.

Gonzalez-Marrero, Ibrahim; Castaneyra-Ruiz, Leandro; Gonzalez-Toledo, Juan M.; Castaneyra-Ruiz, Agustin; de Paz-Carmona, Hector; Castro, Rafael; Hernandez-Fernaud, Juan R.; Castaneyra-Perdomo, Agustin; Carmona-Calero, Emilia M.



Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology.  


The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ((13)C, (15)N, (36)S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed. PMID:23677009

von Bergen, Martin; Jehmlich, Nico; Taubert, Martin; Vogt, Carsten; Bastida, Felipe; Herbst, Florian-Alexander; Schmidt, Frank; Richnow, Hans-Hermann; Seifert, Jana



Short Communication Application of an immunocapillary electrophoresis assay to the detection of abnormal prion protein in brain, spleen and blood specimens from patients with variant Creutzfeldt-Jakob disease  

Microsoft Academic Search

Sensitive and specific detection of abnormal prion protein in blood could provide a diagnostic test or screening assay for animal and human prion diseases. Here, the application of an immunocapillary electrophoresis (ICE) method developed for sheep scrapie to brain, spleen and blood from patients with Creutzfeldt-Jakob disease (CJD) is described. The assay involves organic-solvent extraction, a competitive immunoassay using fluorescently

Paula C. Lourenco; Mary Jo Schmerr; Ian MacGregor; Robert G. Will; James W. Ironside; Mark W. Head


Quantitative Real-Time Imaging of Protein-Protein Interactions by LSPR Detection with Micropatterned Gold Nanoparticles.  


Localized surface plasmon resonance (LSPR) offers powerful means for sensitive label-free detection of protein-protein interactions in a highly multiplexed format. We have here established self-assembly and surface modification of plasmonic nanostructures on solid support suitable for quantitative protein-protein interaction analysis by spectroscopic and microscopic LSPR detection. These architectures were obtained by layer-by-layer assembly via electrostatic attraction. Gold nanoparticles (AuNP) were adsorbed on a biocompatible amine-terminated poly(ethylene glycol) (PEG) polymer brush and further functionalized by poly-l-lysine graft PEG (PLL-PEG) copolymers. Stable yet reversible protein immobilization was achieved via tris(nitrilotriacetic acid) groups incorporated into the PLL-PEG coating. Thus, site-specific immobilization of His-tagged proteins via complexed Ni(II) ions was achieved. Functional protein immobilization on the surface was confirmed by real-time detection of LSPR scattering by reflectance spectroscopy. Association and dissociation rate constants obtained for a reversible protein-protein interaction were in good agreement with the data obtained by other surface-sensitive detection techniques. For spatially resolved detection, AuNP were assembled into micropatterns by means of photolithographic uncaging of surface amines. LSPR imaging of reversible protein-protein interactions was possible in a conventional wide field microscope, yielding detection limits of ?30 protein molecules within a diffraction-limited surface area. PMID:24016060

Bhagawati, Maniraj; You, Changjiang; Piehler, Jacob



Evaluation of three different protocols of protein extraction for Arabidopsis thaliana leaf proteome analysis by two-dimensional electrophoresis  

Microsoft Academic Search

This work was performed to compare three precipitation protocols of protein extraction for 2-DE proteomic analysis using Arabidopsis leaf tissue: TCA–acetone, phenol, and TCA–acetone–phenol. There were no statistically significant differences in protein yield between the three methods. Samples were subjected to 2-DE in the 5 to 8 pH and 14–80 kDa ranges. The TCA–acetone–phenol protocol provided the best results in terms

Ana M. Maldonado; Sira Echevarría-Zomeño; Sylvia Jean-Baptiste; Martha Hernández; Jesús V. Jorrín-Novo



Quantitative protein and fat metabolism in bull calves treated with ß?adrenergic agonist  

Microsoft Academic Search

Protein and energy utilization and quantitative retention of protein, fat and energy was investigated with 12 Red Danish bulls during two subsequent 6 weeks trials (Section A and B) at a mean live weight of 195 and 335 kg respectively. Treatments were control (Group 1) and ß?agonist (L?644, 969) treated animals (Group 2 and 3). ß?agonist supplementation was 5 and

A. Chwalibog; K. Jensen; G. Thorbek



The use of a juvenile hormone binding protein for the quantitative assay of juvenile hormone  

Microsoft Academic Search

The suitability of the haemolymph juvenile hormone binding protein (JHBP) of Locusta migratoria for use in a competition assay for juvenile hormone (JH) III has been investigated, and a simple quantitative assay procedure using this protein has been developed. JHBP partially purified from haemolymph of precocene treated adult locusts gives rapid and stable binding of [3H]10R-JH III, and can be

A. V. Glinka; R. P. Braun; J. P. Edwards; G. R. Wyatt



Distinct and Overlapping Sets of SUMO1 and SUMO2 Target Proteins Revealed by Quantitative Proteomics  

Microsoft Academic Search

The small ubiquitin-like modifier (SUMO) family in verte- brates includes three different family members that are conjugated as post-translational modifications to target proteins. SUMO-2 and -3 are nearly identical but differ substantially from SUMO-1. We used quantitative pro- teomics to investigate the target protein preferences of SUMO-1 and SUMO-2. HeLa cells were established that stably express His6-SUMO-1 or His6-SUMO-2. These

Alfred C. O. Vertegaal; Jens S. Andersen; Stephen C. Ogg; Ronald T. Hay; Matthias Mann; Angus I. Lamond



Antibody response to epitopes of chlamydial major outer membrane proteins on infectious elementary bodies and of the reduced polyacrylamide gel electrophoresis-separated form.  

PubMed Central

Approximately 60% of the outer membrane of chlamydial elementary bodies (EBs) consists of the major outer membrane protein (MOMP) that has structural and metabolic functions. The antigenic properties of MOMPs from mammalian strains of serovars 1 and 2 and an avian strain of Chlamydia psittaci were analyzed. Polyclonal-monospecific antisera (PMAs), one monoclonal antibody (MAb), and polyclonal antisera (PAs) were produced against reduced polyacrylamide gel electrophoresis-separated MOMPs and against infectious EBs. Three PMAs and the MAb, which were induced by reduced polyacrylamide gel electrophoresis-separated MOMPs, reacted strongly in Western blot (immunoblot) assays with MOMPs of serovar 1 and 2 strains as well as with that of the avian strain 6BC, and two of these PMAs reacted weakly (dilution, 1:20) with the MOMP of strain LGV-2. The third PMA and the MAb against the MOMP of the serovar 2 strain did not react with the MOMP of LGV-2. Four PAs were produced against infectious EBs of the serovar 1 strain. One of these PAs reacted with the homologous MOMP and that of the avian strain 6BC but did not recognize MOMPs of other chlamydial strains. Three of the PAs reacted with MOMPs of homologous strains only and failed to recognize MOMPs of avian, serovar 2, and LGV-2 strains. Five PAs induced against infectious EBs of the serovar strain 2 reacted only with the MOMPs of the homologous strains and failed to recognize MOMPs of other strains of chlamydiae. Consequently, MOMPs of C. psittaci strains possess genus-, species-, and serovar-specific epitopes whereby the immune response to serovar-specific epitopes of MOMP predominate when infectious EBs are used for immunization. Images

Baghian, A; Shaffer, L; Storz, J



Label-free quantitative proteomics reveals differentially regulated proteins in experimental gingivitis.  


We investigated the sequential protein expression in gingival crevicular fluid samples during the induction (I) and resolution (R) of experimental gingivitis. Periodontally and systemically healthy volunteers (n = 20) participated in a three-week experimental gingivitis protocol, followed by debridement and two weeks of regular plaque control. Gingival crevicular fluid (GCF) samples were collected at baseline, Day 7, 14, and 21 (induction; I-phase), and at Day 21, 25, 30, and 35 (resolution; R-phase). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for label-free quantitative proteomics was applied. A total of 287 proteins were identified including 254 human, 14 bacterial, 12 fungal, and 7 yeast proteins. Ontology analysis revealed proteins primarily involved in cytoskeletal rearrangements, immune response, antimicrobial function, protein degradation, and DNA binding. There was considerable variation in the number of proteins identified, both among subjects and within subjects across time points. After pooling of samples between subjects at each time point, the levels of 59 proteins in the I-phase and 73 proteins in the R-phase were quantified longitudinally. Our data demonstrate that LC-MS/MS label-free quantitative proteomics is valuable in the assessment of the protein content of the GCF and can facilitate a better understanding of the molecular mechanisms involved in the induction and resolution of plaque-induced gingival inflammation in humans. PMID:23244068

Bostanci, Nagihan; Ramberg, Per; Wahlander, Åsa; Grossman, Jonas; Jönsson, Daniel; Barnes, Virginia Monsul; Papapanou, Panos N



High-Throughput Multiplexed Quantitation of Protein Aggregation and Cytotoxicity in a Huntington's Disease Model  

PubMed Central

A hallmark of Huntington’s disease is the presence of a large polyglutamine expansion in the first exon of the Huntingtin protein and the propensity of protein aggregation by the mutant proteins. Aberrant protein aggregation also occurs in other polyglutamine expansion disorders, as well as in other neurodegenerative diseases including Parkinson’s, Alzheimer’s, and prion diseases. However, the pathophysiological role of these aggregates in the cell death that characterizes the diseases remains unclear. Identification of small molecule probes that modulate protein aggregation and cytotoxicity caused by aggregated proteins may greatly facilitate the studies on pathogenesis of these diseases and potentially lead to development of new therapies. Based on a detergent insoluble property of the Huntingtin protein aggregates, we have developed a homogenous assay to rapidly quantitate the levels of protein aggregates in a cellular model of Huntington’s disease. The protein aggregation assay has also been multiplexed with a protease release assay for the measurement of cytotoxicity resulting from aggregated proteins in the same cells. Through a testing screen of a compound library, we have demonstrated that this multiplexed cytotoxicity and protein aggregation assay has ability to identify active compounds that prevent cell death and/or modulate protein aggregation in cells of the Huntington’s disease model. Therefore, this multiplexed screening approach is also useful for development of high-throughput screening assays for other neurodegenerative diseases involving protein aggregation.

Titus, Steven A; Southall, Noel; Marugan, Juan; Austin, Christopher P; Zheng, Wei



A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.  


To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction. PMID:23871997

Kim, Min Young; Chae, Ji Hyung; Oh, Chang-Ho; Kim, Chul Geun



Detection and Quantitation of T-2 Mycotoxin Using a Simplified Protein Synthesis Inhibition Assay.  

National Technical Information Service (NTIS)

A simple, rapid and sensitive bioassay is described for the detection and quantitation of T-2 mycotoxin using a protein synthesis assay in cultured cells. Increased sensitivity of the cells of the mycotoxin occurs with time up to approximately 60 min. Tim...

W. L. Thompson R. W. Wannemacher



Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells  

Microsoft Academic Search

Green fluorescent protein (GFP) has gained widespread use as a tool to visualize spatial and temporal patterns of gene expression in vivo. However, it is not generally accepted that GFP can also be used as a quantitative reporter of gene expression. We report that GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured

Mark R. Soboleski; Jason Oaks; William P. Halford



Automated Online Sequential Isotope Labeling for Protein Quantitation Applied to Proteasome Tissue-specific Diversity  

Microsoft Academic Search

Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic labeling. To analyze tissue-derived samples, the isotopic labeling can be performed using chemical labeling of the peptides post-digestion. Standard chemical labeling procedures often require many manual sample handling steps, reduc- ing the accuracy of measurements. Here, we

Reinout Raijmakers; Celia R. Berkers; Annemieke de Jong; Huib Ovaa; Albert J. R. Heck; Shabaz Mohammed



Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae  

PubMed Central

The cell wall of yeast consists of an internal skeletal layer and an external layer of glycoproteins covalently linked to the stress-bearing polysaccharides. The cell wall protein (CWP) population consists of over 20 different proteins, and may vary in composition. We present two complementary methods for quantifying CWPs, based on isobaric tagging and tandem MS: (1) absolute quantitation of individual CWPs, allowing estimation of surface densities; and (2) relative quantitation of CWPs, allowing monitoring of the dynamics of the CWP population. For absolute quantitation, we selected a representative group of five proteins (Cwp1p, Crh1p, Scw4p, Gas1p, and Ecm33p), which had 67 × 103, 44 × 103, 38 × 103, 11 × 103 and 6.5 × 103 of wall-bound copies per cell, respectively. As Cwp1p is predominantly incorporated in the birth scar, this corresponds to a protein density of c. 22 × 103 copies ?m?2. For relative quantitation, we compared wild-type cells to gas1? cells, in which the cell wall integrity pathway is constitutively activated. The levels of Crh1p, Crh2p, Ecm33p, Gas5p, Pst1p and Pir3p increased about three- to fivefold, whereas the level of Scw4p was significantly decreased. We propose that our methods are widely applicable to other fungi.

Yin, Qing Yuan; de Groot, Piet W J; de Jong, Luitzen; Klis, Frans M; De Koster, Chris G



Novel Proteins, Putative Membrane Transporters, and an Integrated Metabolic Network Are Revealed by Quantitative Proteomic Analysis of Arabidopsis Cell Culture Peroxisomes1[W][OA  

PubMed Central

Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of these important functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomes and their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis (Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained using two new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed density gradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, we used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomal proteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core peroxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to be peroxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likely to be components in peroxisome biogenesis, ?-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration, and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potential roles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a tight integration of functions and highlights specific metabolite nodes that most probably represent entry and exit metabolites that could require transport across the peroxisomal membrane.

Eubel, Holger; Meyer, Etienne H.; Taylor, Nicolas L.; Bussell, John D.; O'Toole, Nicholas; Heazlewood, Joshua L.; Castleden, Ian; Small, Ian D.; Smith, Steven M.; Millar, A. Harvey



Identification of Pseudomonas proteins coordinately induced by acidic amino acids and their amides: a two-dimensional electrophoresis study.  


The acidic amino acids (Asp, Glu) and their amides (Asn, Gln) are excellent growth substrates for many pseudomonads. This paper presents proteomics data indicating that growth of Pseudomonas fluorescens ATCC 13525 and Pseudomonas putida KT2440 on these amino acids as sole source of carbon and nitrogen leads to the induction of a defined set of proteins. Using mass spectrometry and N-terminal sequencing, a number of these proteins were identified as enzymes and transporters involved in amino acid uptake and metabolism. Most of them depended on the alternative sigma factor sigma(54) for expression and were subject to strong carbon catabolite repression by glucose and citrate cycle intermediates. For a subset of the identified proteins, the observed regulatory effects were independently confirmed by RT-PCR. The authors propose that the respective genes (together with others still to be identified) make up a regulon that mediates uptake and utilization of the abovementioned amino acids. PMID:14523123

Sonawane, Avinash; Klöppner, Ute; Hövel, Sven; Völker, Uwe; Röhm, Klaus-Heinrich



Optimization of an Efficient Protein Extraction Protocol Compatible with Two-Dimensional Electrophoresis and Mass Spectrometry from Recalcitrant Phenolic Rich Roots of Chickpea (Cicer arietinum L.).  


Two-dimensional electrophoresis and mass spectrometry are undoubtedly two essential tools popularly used in proteomic analyses. Utilization of these techniques however largely depends on efficient and optimized sample preparation, regarded as one of the most crucial steps for recovering maximum amount of reliable information. The present study highlights the optimization of an effective and efficient protocol, capable of extraction of root proteins from recalcitrant phenolic rich tissues of chickpea. The widely applicable TCA-acetone and phenol-based methods have been comparatively evaluated, amongst which the latter appeared to be better suited for the sample. The phenol extraction-based method further complemented with sodium dodecyl sulphate (SDS) and pulsatory treatments proved to be the most suitable method represented by greatest spot number, good resolution, and spot intensities. All the randomly selected spots showed successful identification when subjected to further downstream MALDI-TOF and MS/MS analyses. Hence, the information obtained collectively proposes the present protein extraction protocol to be an effective one that could be applicable for recalcitrant leguminous root samples. PMID:23193474

Chatterjee, Moniya; Gupta, Sumanti; Bhar, Anirban; Das, Sampa



Towards comprehensive analysis of protein family quantitative stability-flexibility relationships using homology models.  


The Distance Constraint Model (DCM) is a computational modeling scheme that uniquely integrates thermodynamic and mechanical descriptions of protein structure. As such, quantitative stability-flexibility relationships (QSFR) that describe the interrelationships of thermodynamics and mechanics can be quickly computed. Using comparative QSFR analyses, we have previously investigated these relationships across a small number of protein orthologs, ranging from two to a dozen [1, 2]. However, our ultimate goal is provide a comprehensive analysis of whole protein families, which requires consideration of many more structures. To that end, we have developed homology modeling and assessment protocols so that we can robustly calculate QSFR properties for proteins without experimentally derived structures. The approach, which is presented here, starts from a large ensemble of potential homology models and uses a clustering algorithm to identify the best models, thus paving the way for a comprehensive QSFR analysis across hundreds of proteins in a protein family. PMID:24061925

Verma, Deeptak; Guo, Jun-Tao; Jacobs, Donald J; Livesay, Dennis R



Identification of Mycobacterium fortuitum, Mycobacterium abscessus, and Mycobacterium borstelense by Polyacrylamide Gel Electrophoresis of Their Cell Proteins  

Microsoft Academic Search

Strains of M. fortuitum, M. abscessus, and M. borstelense showed different polyacrylamide gel electrophoretic patterns of their cell proteins. M. fortuitum strains could easily be distinguished from those of M. abscessus and M. borstelense, and they appear to belong to a single homogeneous group. M. abscessus and M. borstelense gels showed similar patterns, thus tending to confirm recent suggestions that



Distinction Between Cereal Genotypes Based on the Protein and DNA Composition of the Grain by Capillary Electrophoresis  

Microsoft Academic Search

Cereal grains are widely used for human food and animal feed throughout the world. Distinction between the various genotypes of any cereal species is important to segregate grains according to utilization type. DNA analysis indicates genotype (variety), whereas protein composition provides information about both variety and likely processing properties, reflecting the contributions of both genotype and growth\\/storage conditions. Standard methods

O. Kaisoon; S. Siriamornpun; N. Meeso



Partitioning of limiting protein and energy in the growing pig: testing quantitative rules against experimental data.  


Literature solutions to the problem of protein and energy partitioning in the growing pig are quantitatively examined. Possible effects of live weight, genotype and food composition on the marginal response in protein retention to protein and energy intakes, on protein and energy-limiting foods are quantified. No evidence was found that the marginal response in protein retention to ideal protein supply, when protein intake is limiting, is affected by live weight, genotype or environmental temperature. There was good evidence that live weight does not affect the marginal response in protein retention to energy intake when protein intake is not limiting. Limited data for different genotypes suggested no effects on this response. A general quantitative partitioning rule is proposed that has two key parameters; e(p)* (the maximum marginal efficiency for retaining the first limiting amino acid) and R* (the maximum value of R, the energy to protein ratio of the food, MJ metabolisable energy (ME)/kg digestible crude protein (DCP), when e(p)* is just achieved). When Rprotein is (e(p)*/R*) x R. The value of e(p)* was determined to be 0.763 (SE 0.0130). There was no good experimental evidence that e(p)* is different for different amino acids. The best estimate of R* was 67.9 (SE 1.65) MJ ME/kg DCP. Live weight, genotype and temperature did not affect the values of either parameter. A more general understanding of partitioning, including the effects of 'stressors' such as disease, may be achieved by using the preferred rule as a starting point. PMID:15788115

Sandberg, Fredrik B; Emmans, Gerry C; Kyriazakis, Ilias



Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair.  


Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein-protein interaction and even protein modifications in living cells. Here, we analyze the E(0)GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro. The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R(0) = 51 A), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E(0)GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo. Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo) reveals that this new pair can be used for very effective quantitative FRET imaging. PMID:18764891

Albertazzi, Lorenzo; Arosio, Daniele; Marchetti, Laura; Ricci, Fernanda; Beltram, Fabio



Profiling excretory\\/secretory proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry  

Microsoft Academic Search

Infection of mammalian skeletal muscle with the intracellular parasite Trichinella spiralis results in profound alterations in the host cell and a realignment of host cell gene expression. The role of parasite excretory\\/secretory (E\\/S) products in mediating these effects is unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, we have used two-dimensional

Mark W. Robinson; Daniel C. Gare; Bernadette Connolly



Use of SDS to Extract Sorghum and Maize Proteins for Free Zone Capillary Electrophoresis (FZCE) Analysis 1  

Microsoft Academic Search

Cereal Chem. 78(1):84-87 Two different extraction methods for extracting sorghum ( Sorghum bicolor L. Moench.) storage proteins for free zone capillary electr ophor- esis (FZCE) analysis were compared. A traditional solvent based on 60% t-butanol was compared with a pH 10 borate buffer containing the anionic detergent SDS followed by precipitation of nonkafirins using 60% t-buta- nol. FZCE analysis of

S. R. Bean; C. Hicks; M. Tuinstra; G. L. Lookhart



Identification of Pseudomonas proteins coordinately induced by acidic amino acids and their amides: a two-dimensional electrophoresis  

Microsoft Academic Search

The acidic amino acids (Asp, Glu) and their amides (Asn, Gln) are excellent growth substrates for many pseudomonads. This paper presents proteomics data indicating that growth of Pseudomonas fluorescens ATCC 13525 and Pseudomonas putida KT2440 on these amino acidsas solesourceofcarbonandnitrogen leadstotheinduction ofadefinedsetofproteins.Using mass spectrometry and N-terminal sequencing, a number of these proteins were identified as enzymes and transporters involved in

Avinash Sonawane; Ute Kloppner; Sven Hovel; Uwe Volker; Klaus-Heinrich Roh


Microgel electrophoresis: sensitivity, mechanisms, and DNA electrostretching.  


Based on the treatment of microgels to remove proteins, we speculate that proteins may be bound to DNA in the microgels even after electrophoresis. We speculate that some DNA single-strand breaks may be a reflection of these protein-DNA complexes. We suggest methods to limit such artifacts, and present data demonstrating a lymphocyte DNA double-strand break sensitivity of 12.5 rads and day-to-day reproducibility of microgel electrophoresis using these principles. Extending these principles, we describe DNA behavior during alkaline and neutral microgel electrophoresis based on observations of the stained DNA and its migration patterns. During microgel electrophoresis, individual DNA molecules behave as if anchored at one end while the other end is free to migrate in response to the electric field. We capitalize on this behavior by developing a neutral microgel method to stretch chromosomes. PMID:9088349

Singh, N P; Stephens, R E



Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions.  


We demonstrate quantitative multicolor three-dimensional (3D) subdiffraction imaging of the structural arrangement of fluorescent protein fusions in living Caulobacter crescentus bacteria. Given single-molecule localization precisions of 20-40 nm, a flexible locally weighted image registration algorithm is critical to accurately combine the super-resolution data with <10 nm error. Surface-relief dielectric phase masks implement a double-helix response at two wavelengths to distinguish two different fluorescent labels and to quantitatively and precisely localize them relative to each other in 3D. PMID:23414562

Gahlmann, Andreas; Ptacin, Jerod L; Grover, Ginni; Quirin, Sean; von Diezmann, Alexander R S; Lee, Marissa K; Backlund, Mikael P; Shapiro, Lucy; Piestun, Rafael; Moerner, W E



Complementary Analysis of the Mycobacterium tuberculosis Proteome by Two-dimensional Electrophoresis and Isotope-coded Affinity Tag Technology  

Microsoft Academic Search

Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantifica- tion of proteins in a complex mixture with mass spectro- metric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS\\/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobac- terium tuberculosis,

Frank Schmidt; Samuel Donahoe; Kristine Hagens; Jens Mattow; Ulrich E. Schaible; Stefan H. E. Kaufmann; Ruedi Aebersold; Peter R. Jungblut



Quantitative Fluorescent Labeling of Aldehyde-Tagged Proteins for Single-Molecule Imaging  

PubMed Central

A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescent labeling with high efficiency, specificity, and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ~100% efficiency while maintaining the biological function. We demonstrate that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. We further show the unique power of our method in a series of single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor.

Shi, Xinghua; Jung, Yonil; Lin, Li-Jung; Liu, Cheng; Wu, Cong; Cann, Isaac K. O.; Ha, Taekjip