Sample records for quantitative protein electrophoresis

  1. technical manual protein electrophoresis

    E-print Network

    Kirschner, Marc W.

    . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Chapter 2 Polyacrylamide Gel Electrophoresis.2 Separating proteins on the basis of molecular weight: SDS gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.5 Native gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 2

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

    NASA Astrophysics Data System (ADS)

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

    2010-04-01

    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.

  3. Protein electrophoresis - serum

    MedlinePLUS

    This lab test measures the types of protein in the fluid (serum) part of a blood sample. Other electrophoresis tests that measure proteins in the serum include: Immunoelectrophoresis Immunofixation Globulin electrophoresis

  4. Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis.

    PubMed

    Sachon, E; Mohammed, S; Bache, N; Jensen, O N

    2006-01-01

    Polyacrylamide gel electrophoresis is widely used for protein separation and it is frequently the final step in protein purification in biochemistry and proteomics. Using a commercially available amine-reactive isobaric tagging reagent (iTRAQ) and mass spectrometry we obtained reproducible, quantitative data from peptides derived by tryptic in-gel digestion of proteins and phosphoproteins. The protocol combines optimized reaction conditions, miniaturized peptide handling techniques and tandem mass spectrometry to quantify low- to sub-picomole amounts of (phospho)proteins that were isolated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immobilized metal affinity chromatography (FeIII-IMAC) was efficient for removal of excess reagents and for enrichment of derivatized phosphopeptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Phosphopeptide abundance was determined by liquid chromatography/tandem mass (LC/MS/MS) using either MALDI time-of-flight/time-of-flight (TOF/TOF) MS/MS or electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS/MS instruments. Chemically labeled isobaric phosphopeptides, differing only by the position of the phosphate group, were distinguished and characterized by LC/MS/MS based on their LC elution profile and distinct MS/MS spectra. We expect this quantitative mass spectrometry method to be suitable for systematic, comparative analysis of molecular variants of proteins isolated by gel electrophoresis. PMID:16521170

  5. Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis.

    PubMed

    Kralj, Jason G; Munson, Matthew S; Ross, David

    2014-07-01

    We investigated the ability of gradient elution moving boundary electrophoresis (GEMBE) with capacitively coupled contactless conductivity detection (C(4) D) to assay total protein concentration using the bicinchoninic acid (BCA) reaction. We chose this format because GEMBE-C(4) D behaves as a concentration dependent detection system, unlike optical methods that also rely on pathlength (due to Beer's law). This system tolerates proteins well compared with other capillary electrophoretic methods, allowing the capillary to be reused without coatings or additional hydroxide wash steps. The typical reaction protocol was modified by reducing the pH slightly from 11.25 to 9.4, which enabled elimination of tartrate from the reagents. We estimated that copper (I) could be detected at approximately 3.0 ?mol/L, which agrees with similar GEMBE and CZE systems utilizing C(4) D. Under conditions similar to the BCA "micro method" assay, we determined the LOD for three common proteins (insulin, BSA, and bovine gamma globulin) and found that they agree well with the existing spectroscopic detection methods. Further, we investigated how long reaction times impact the LOD and found that the conversion was proportional to log(time). This indicated that little sensitivity is gained by extending the reaction past 1 h. Hence, GEMBE provides an alternative platform for total protein assays while maintaining the excellent sensitivity of the optical-based methods. PMID:24648165

  6. Protein Electrophoresis/Immunofixation Electrophoresis

    MedlinePLUS

    ... Back to top 3. What are free light chains and how are they related to immunoglobulins? Immunoglobulins are molecules composed of four protein chains: two identical light chains, either kappa or lambda ...

  7. Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

    PubMed Central

    2014-01-01

    Background Certain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated. Results Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction. Conclusions This is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the analysis revealed additional layers of complexity to the wheat flour proteome that should be considered when evaluating quantitative 2-DE data. PMID:24517725

  8. Quantitative analysis of two-dimensional gel electrophoresis protein patterns: a method for studying genetic relationships among Globodera pallida populations.

    PubMed

    Fullaondo, A; Vicario, A; Aguirre, A; Barrena, I; Salazar, A

    2001-09-01

    A method based in two-dimensional protein gel electrophoresis has been developed in order to improve the analysis of genetic relationships among populations of Globodera. It has been used to estimate genetic divergence among nine Globodera pallida nematode populations. Sixty-one anonymous polypeptide spots were resolved using silver-stained high-resolution 2D gels and they were quantified in each population to establish genetic variation among G. pallida populations. The results of this analysis were compared with those obtained after a study of allelic frequency variation, which was carried out using seven previously described loci. Genetic distances among populations were calculated by means of both studies, the quantitative analysis and the allelic frequency variation, and phylogenetic trees were constructed for each type of analysis. A correlation analysis between the two distance matrices was carried out and a bootstrap analysis was performed to determine the strength of the clusters obtained with each method. The results obtained support the idea that quantitative protein analysis can be successfully applied to phylogenetic analysis of G. pallida populations. PMID:11737273

  9. Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication and further separated by size exclusion chromatography into monomeric and polymeric fractions. Proteins in each fraction were analyzed by quantitative two-dimensional gel...

  10. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    SciTech Connect

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    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.

  11. Quantitation of Caseins and Whey Proteins of Processed Milks and Whey Protein Concentrates, Application of Gel Electrophoresis, and Comparison with Harland-Ashworth Procedure

    Microsoft Academic Search

    Jay J. Basch; Frederic W. Douglas Jr.; Lisa G. Procino; V. H. Holsinger; Harold M. Farrell Jr.

    1985-01-01

    Alternate methods for quantitation of caseins and whey proteins in milk pro- ducts were investigated. The Harland- Ashworth and Leighton procedures, which are used for routine determinations of soluble whey proteins in milk, could not be adapted satisfactorily to quantitation of whey protein in blends of nonfat dry milk solids and whey protein concentrates because of problems of precipitation techniques.

  12. Human lymphocyte polymorphisms detected by quantitative two-dimensional electrophoresis

    SciTech Connect

    Goldman, D.; Merril, C.R.

    1983-09-01

    A survey of 186 soluble lymphocyte proteins for genetic polymorphism was carried out utilizing two-dimensional electrophoresis of /sup 14/C-labeled phytohemagglutinin (PHA)-stimulated human lymphocyte proteins. Nineteen of these proteins exhibited positional variation consistent with independent genetic polymorphism in a primary sample of 28 individuals. Each of these polymorphisms was characterized by quantitative gene-dosage dependence insofar as the heterozygous phenotype expressed approximately 50% of each allelic gene product as was seen in homozygotes. Patterns observed were also identical in monozygotic twins, replicate samples, and replicate gels. The three expected phenotypes (two homozygotes and a heterozygote) were observed in each of 10 of these polymorphisms while the remaining nine had one of the homozygous classes absent. The presence of the three phenotypes, the demonstration of gene-dosage dependence, and our own and previous pedigree analysis of certain of these polymorphisms supports the genetic basis of these variants. Based on this data, the frequency of polymorphic loci for man is: P . 19/186 . .102, and the average heterozygosity is .024. This estimate is approximately 1/3 to 1/2 the rate of polymorphism previously estimated for man in other studies using one-dimensional electrophoresis of isozyme loci. The newly described polymorphisms and others which should be detectable in larger protein surveys with two-dimensional electrophoresis hold promise as genetic markers of the human genome for use in gene mapping and pedigree analyses.

  13. SDS capillary gel electrophoresis of proteins in microfabricated channels

    PubMed Central

    Yao, Shao; Anex, Deon S.; Caldwell, W. Brett; Arnold, Don W.; Smith, Katherine B.; Schultz, Peter G.

    1999-01-01

    Analysis of variations in the concentrations or structures of biomolecules (e.g., mRNAs, proteins, peptides, natural products) that occur either naturally or in response to environmental or genetic perturbations can provide important insight into complex biological processes. Many biological samples are mixtures that require a separation step before quantitation of variations in the individual components. Two-dimensional denaturing gel electrophoresis has been used very effectively to separate complex mixtures of proteins, but it is time consuming and requires considerable amounts of sample. Microchannel-based separations have proven very effective in rapidly separating small amounts of nucleic acids; more recently, isoelectric focusing of proteins also has been adapted to the microchannel format. Here, we describe microchannel-based SDS capillary gel electrophoresis of proteins and demonstrate the speed and high resolution it provides. This development is an important step toward the miniaturization and integration of multidimensional and array separation methods for complex protein mixtures. PMID:10318890

  14. Quantitative Analysis of Human Milk Oligosaccharides by Capillary Electrophoresis

    Microsoft Academic Search

    David S. Newburg; Zuojun Shen; Christopher D. Warren

    Human milk oligosaccharides may have important biological activities. 1 We developed a sensitive, convenient, quantitative method for the routine study of sialylated (acidic, negatively charged) oligosaccharides in large numbers of milk samples. Capillary electrophoresis (CE) with detection at 205 nm was sensitive to the femtomole level and could resolve and quantify nine acidic oligosaccharides in milk, ranging from tri- to

  15. Serum protein electrophoresis in 147 dogs

    Microsoft Academic Search

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

    2011-01-01

    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

  16. Extensions of Gel Electrophoresis with Proteins

    NSDL National Science Digital Library

    Mr. Brian McClain (Amos P. Godby High School)

    2006-04-01

    This inquiry activity is intended to help familiarize students with the procedure of agarose electrophoresis and to make them aware of the types of proteins within tissue samples. This inquiry activity was developed by a K-12 science teacher in the American Physiological SocietyÂ?s 2006 Frontiers in Physiology Program. The NSES Standards addressed by this activity are current as of the year of development. For more information on the Frontiers in Physiology Program, please visit www.frontiersinphys.org.

  17. Using Gel Electrophoresis To Illustrate Protein Diversity and Isoelectric Point.

    ERIC Educational Resources Information Center

    Browning, Mark; Vanable, Joseph

    2002-01-01

    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)

  18. Seperation of proteins using cetyltrimethylammonium bromide discontinuous gel electrophoresis

    Microsoft Academic Search

    Robert E. Akins; Rocky S. Tuan

    1994-01-01

    The gel electrophoresis system presented here allows the separation of proteins with the concomitant retention of detectable\\u000a native activities. The system, referred to as CAT gel electrophoresis. uses the detergent cetyltrimethylammonium bromide in\\u000a combination with a discontinuous gel matrix to resolve protein mixtures into discrete bands. Many proteins retain detectable\\u000a levels of native activity after CAT electrophoresis, and gel bands

  19. Viral quantitative capillary electrophoresis for counting intact viruses.

    PubMed

    Mironov, Gleb G; Chechik, Alexey V; Ozer, Rachel; Bell, John C; Berezovski, Maxim V

    2011-07-01

    The quantification of a virus plays an important role in vaccine development, clinical diagnostics, and environmental contamination assays. In all these cases, it is essential to calculate the concentration or number of intact virus particles (ivp) and estimate the degree of degradation and contamination of virus samples. In this work, we propose a cost-efficient, robust method for the quantification and characterization of intact viruses based on capillary zone electrophoresis. This separation method is demonstrated on vaccinia virus (VV) with oncolytic properties. After virus sample preparation, the solution contains intact VV as well as broken viruses and residual DNA from the host cell used for preparation. Regulatory requirements limit the amount of the host cell DNA that can be present in vaccines or human therapeutics. We apply capillary electrophoresis to separate intact virus particles and the residual DNA and to measure the level of virus contamination with DNA impurities. Intercalating YOYO-1 dye is used to detect the encapsulated and free DNA by laser-induced fluorescence. After soft lysis of VV with proteinase K, all encapsulated DNA is dissolved to the free DNA. The change in peak areas and a DNA calibration curve help determine the initial concentration of intact viruses. This viral quantitative capillary electrophoresis (Viral qCE) is able to quantify the oncolytic vaccinia virus in the range of 10(6) to 10(12) ivp/mL. PMID:21599011

  20. Quantification of Polyacrylamide Gel Electrophoresis for Analysis of Whey Proteins

    Microsoft Academic Search

    D. F. Darling; D. W. Butcher

    1976-01-01

    Polyacrylamide gel electrophoresis of whey proteins has been quantified by standardization of the separation and staining procedure. During each electro- phoresis experiment, a standard solution of whey proteins was separated and stained under the same conditions as the test material. In this way, proteins in the standard solution were subjected to iden- tical processing conditions as the test samples. Densitometric

  1. Application of capillary zone electrophoresis and reversed-phase high-performance liquid chromatography in the biopharmaceutical industry for the quantitative analysis of the monosaccharides released from a highly glycosylated therapeutic protein.

    PubMed

    Racaityte, K; Kiessig, S; Kálmán, F

    2005-06-24

    Two assays for the quantitative determination of the neutral and amino-monosaccharides attached to a therapeutic glycoprotein were developed using capillary zone electrophoresis (CZE) and RP-HPLC. These assays meet the strict batch release requirements of the quality control in biopharmaceutical industry. The monosaccharides were released from the glycoprotein by hydrolysis with 2N trifluoroacetic acid. In the CZE assay the monosaccharides were reacetylated prior to derivatization with 8-aminopyrenesulfonic acid (APTS), reacetylation in the glycoprotein matrix was investigated in detail. The RP-HPLC method used pre-column derivatization with anthranilic acid in methanol-acetate-borate reaction medium; reacetylation was not necessary. However, epimerization of the different monosaccharides was observed and studied in detail. For the quantitative assay, separation of the amino-monosaccharide epimers had to be developed. The HPLC assay was validated. PMID:16038323

  2. Analysis of protein glycation using fluorescent phenylboronate gel electrophoresis

    PubMed Central

    Pereira Morais, Marta P.; Marshall, Dominic; Flower, Stephen E.; Caunt, Christopher J.; James, Tony D.; Williams, Robert J.; Waterfield, Nicholas R.; van den Elsen, Jean M. H.

    2013-01-01

    Glycated proteins are important biomarkers for age-related disorders, however their analysis is challenging because of the complexity of the protein-carbohydrate adducts. Here we report a method that enables the detection and identification of individual glycated proteins in complex samples using fluorescent boronic acids in gel electrophoresis. Using this method we identified glycated proteins in human serum, insect hemolymph and mouse brain homogenates, confirming this technique as a powerful proteomics tool that can be used for the identification of potential disease biomarkers. PMID:23531746

  3. Human muscle proteins: analysis by two-dimensional electrophoresis

    SciTech Connect

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

    1983-09-01

    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.

  4. Monitoring intracellular protein profiles with two-dimensional gel electrophoresis.

    PubMed

    Dykstra, K H; Wang, H Y

    1992-01-01

    Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is a method of separating complex protein mixtures, such as whole cell extracts, on the basis of protein isoelectric point and molecular weight. In bioprocess engineering, conventional 2D PAGE has tremendous potential to yield detailed information on the intracellular effect of various process conditions. It has been used in our work to examine global intracellular changes occurring in a typical cycloheximide fermentation and to look at the feedback regulatory behavior of cycloheximide biosynthesis. Application of the technique for bioprocess monitoring will require that the time necessary for preparation of a 2D electropherogram be substantially shortened. This may be accomplished by performing the separation on a miniature scale or eventually by use of capillary electrophoresis for one or more of the separations. Advantages and disadvantages of these two approaches are discussed. PMID:1368457

  5. Protein Separation by Capillary Gel Electrophoresis: A Review

    PubMed Central

    Zhu, Zaifang; Lu, Joann J.; Liu, Shaorong

    2011-01-01

    Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples. PMID:22122927

  6. Procedures for two-dimensional electrophoresis of proteins

    SciTech Connect

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

    1996-10-01

    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.

  7. Capillary Electrophoresis-Based Immunoassays: Principles & Quantitative Applications

    PubMed Central

    Moser, Annette C.; Hage, David S.

    2013-01-01

    The use of capillary electrophoresis as a tool to conduct immunoassays has been an area of increasing interest over the last decade. This approach combines the efficiency, small sample requirements, and relatively high speed of CE with the selectivity of antibodies as binding agents. This review examines the various assay formats and detection modes that have been reported for these assays, along with some representative applications. Most CE immunoassays in the past have employed homogeneous methods in which the sample and reagents are allowed to react in solution. These homogeneous methods have been conducted as both competitive binding immunoassays and as non-competitive binding immunoassays. Fluorescent labels are most commonly used for detection in these assays, but enzyme labels have also been utilized for such work. Some additional work has been performed in CE immunoassays with heterogeneous methods in which either antibodies or an analog of the analyte is immobilized to a solid support. These heterogeneous methods can be used for the selective isolation of analytes prior to their separation by CE or to remove a given species from a sample/reagent mixture prior to analysis by CE. These CE immunoassays can be used with a variety of detection modes, such as fluorescence, UV/visible absorbance, chemiluminescence, electrochemical measurements, mass spectrometry, and surface plasmon resonance. PMID:18646279

  8. Method for quantitating cholesterol in subfractions of serum lipoproteins separated by gradient gel electrophoresis

    Microsoft Academic Search

    M.-L. Cheng; C. M. Kammerer; W. F. Lowe; B. Dyke; J. L. VandeBerg

    1988-01-01

    Extensive heterogeneity in particle size distribution of serum lipoproteins of baboons was resolved by a procedure that combined\\u000a Sudan black B prestaining, polyacrylamide gradient gel electrophoresis (GGE), and quantitative densitometry. Each densitometric\\u000a scan represented a continuous distribution of the relative amount of cholesterol in a serum sample, as a function of the lipoprotein\\u000a particle size. For analytical purposes, each scan

  9. Quantitative capillary electrophoresis determination of oversulfated chondroitin sulfate as a contaminant in heparin preparations

    Microsoft Academic Search

    Nicola Volpi; Francesca Maccari; Robert J. Linhardt

    2009-01-01

    A simple, accurate, and robust quantitative capillary electrophoresis (CE) method for the determination of oversulfated chondroitin sulfate (OSCS) as a contaminant in heparin (Hep) preparations is described. After degradation of the polysaccharides by acidic hydrolysis, the hexosamines produced (i.e., GlcN from Hep and GalN from OSCS) were derivatized with anthranilic acid (AA) and separated by means of CE in approximately

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

    SciTech Connect

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

    1995-04-01

    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.

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

    ERIC Educational Resources Information Center

    Atkins, Thomas

    1991-01-01

    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)

  12. QUALITATIVE AND QUANTITATIVE CHANGES IN PROTEINS IN ACER PLATANOIDES L. SEEDS DURING MATURATION

    Microsoft Academic Search

    ANDRZEJ KALINOWSKI

    2003-01-01

    Maturation of Norway maple (Acer platanoides L.) seeds produces deep physiological dormancy and resistance to desiccation. This study used two-dimensional electrophoresis to investigate the protein products of genes activated during the complex developmental process of maturation. Qualitative and quantitative changes in protein composition during maturation were tracked in this species. The most intensive changes in protein content appeared at the

  13. Identification of isolate-specific sporozoite proteins of Cryptosporidium parvum by two-dimensional gel electrophoresis.

    PubMed Central

    Mead, J R; Humphreys, R C; Sammons, D W; Sterling, C R

    1990-01-01

    Five isolates of Cryptosporidium parvum collected from human, horse, and calf sources were compared for differences in sporozoite protein patterns by using two-dimensional gel electrophoresis. Silver-stained two-dimensional gels contained over 300 protein spots from detergent-solubilized sporozoites. A distinguishing 106-kilodalton peptide that shifted in isoelectric point was detected in four of the five isolates. Computerized two-dimensional gel analysis was performed to obtain objective quantitation of the pI shift. Three of these four isolates could be differentiated from one other by the pI shift in this peptide. The fifth isolate was distinguished by the absence of the 106-kilodalton peptide and the presence of a 40-kilodalton peptide that was not observed in any other isolate. Images PMID:2365452

  14. Analysis of the origin of Johnson grass, Sorghum halepense (Linn.), by protein electrophoresis

    E-print Network

    Collier, Glen Eldon

    1971-01-01

    of Johnson grass was investigated by means of protein slectrophorssis. Insoluble PUP snd a reducing agent, such as Cleland's reagent, were found to be required in the extraction buffer to insure the recovery of active enzymes from sorghum grain extracts.... MATERIALS AND METHODS Plant Materials. Extraction Electrophoresis. RESULTS. Extraction. Electrophoresis DISCUSSION. Extraction Electrophoresis. SUMMARY AND CONCLUSION. LITERATURE CITED. VITA. iv vi 9 9 13 15 15 28 36 36 38 41 42 46...

  15. Evaluation of serum protein separation by capillary electrophoresis: prospective analysis of 1000 specimens

    Microsoft Academic Search

    Margaret A. Jenkins; Elena Kulinskaya; Helen D. Martin; Michael D. Guerin

    1995-01-01

    To critically assess the method of capillary electrophoresis (CE) we examined 1000 prospective serum samples submitted for protein electrophoresis by both high-resolution agarose gel electrophoresis (HRAGE) and CE. CE was performed using a 72 cm (50 cm to detector) × 50 ?m I.D. fused-silica capillary with detection of absorbance at 200 nm. The 1000 samples examined contained 362 monoclonal paraproteins

  16. ESTIMATION OF THIN PLATE SPLINE WARP PARAMETERS FROM PROTEIN SPOT POSITIONS IN 2D ELECTROPHORESIS GELS

    E-print Network

    - mation and a Thin Plate Spline transformation in the reg- istration of 2D gel electrophoresis imagesESTIMATION OF THIN PLATE SPLINE WARP PARAMETERS FROM PROTEIN SPOT POSITIONS IN 2D ELECTROPHORESIS GELS Lars Pedersen Informatics and Mathematical Modelling Richard Petersens Plads, Building 321

  17. Comparison of capillary electrophoresis with traditional methods to analyse bovine whey proteins

    Microsoft Academic Search

    Nicki M. Kinghorn; Carmen S. Norris; Geoff R. Paterson; Don E. Otter

    1995-01-01

    The separation of the four major whey proteins by sodium dodecyl sulphate (SDS)-capillary gel electrophoresis (CGE) is described. Whilst commercially purified whey proteins could be analysed using the recommended protocol, the more complex nature of an acid whey and a reconstituted whey protein concentrate (WPC) powder necessitated considerable refinement of the CGE sample buffer. Individual whey proteins in the acid

  18. Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane.

    PubMed

    Muramoto, K; Tanaka, H; Kimura, S; Kubo, K; Yoshihara, S; Yokoyama, M; Yoshida, Y; Endo, M

    1989-07-01

    Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms. PMID:2809069

  19. Quantitative prediction of enantioseparation using ?-cyclodextrin derivatives as chiral selectors in capillary electrophoresis.

    PubMed

    Guo, Xin; Wang, Zhiqiang; Zuo, Lihua; Zhou, Zhixu; Guo, Xingjie; Sun, Tiemin

    2014-12-21

    ?-Cyclodextrin derivatives as chiral selectors are becoming increasingly important for enantioseparations in capillary electrophoresis (CE). Nevertheless, there are some enormous challenges in choosing effective selectors from a variety of compounds, and up to now no systematic quantitative studies for predicting the possibility of enantiomeric separation before CE experiments have been reported. In this paper, in order to resolve previous confusions, we investigated the enantioseparations of ten chiral drugs using a method of combining experiments with theoretical calculations. MMFF, PM3, DFT and ONIOM2 methods were simultaneously utilized during the course of our computer simulations. The results indicated that a specific value of greater than or approximately equal to 6 kJ mol(-1) for the interaction energy difference (??E) between a pair of enantiomers with a selector is required in order to achieve enantiomeric separation. This discovery offers a meaningful reference to predict enantiomeric separations, so as to design and synthesize some more efficient chiral selectors. PMID:25346954

  20. THERMAL DETECTION OF DNA AND PROTEINS DURING GEL ELECTROPHORESIS

    SciTech Connect

    R. JOHNSTON

    2000-08-01

    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.

  1. Quantification of the major bovine whey proteins using capillary zone electrophoresis

    Microsoft Academic Search

    Nicki M. Kinghorn; Geoff R. Paterson; Don E. Otter

    1996-01-01

    Bovine whey comprises four major protein groups [?-lactalbumin, ?-lactoglobulin variants A and B, bovine serum albumin and immunoglobulin (specifically IgG) which have a diverse range of molecular masses, pI values, number of phenotypic variants and subunit compositions. The development of a capillary zone electrophoresis method to separate these whey proteins is described. Initially separation of the individual whey proteins was

  2. SEPARATION OF WATER SOLUBLE PROTEINS FROM CEREALS BY FREE ZONE CAPILLARY ELECTROPHORESIS (FZCE)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most research concerning grain proteins has concentrated upon the gluten storage proteins. The albumins and globulins are the water and salt soluble proteins that contain biologically active enzymes and enzyme inhibitors. A Free Zone Capillary electrophoresis method was developed to separate these p...

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

    Microsoft Academic Search

    Ari Helenius; Kai Simons

    1977-01-01

    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

  4. Influence of Proteolysis of Milk on the Whey Protein to Total Protein Ratio as Determined by Capillary Electrophoresis

    Microsoft Academic Search

    B. Miralles; M. Ramos; L. Amigo

    2003-01-01

    Capillary electrophoresis (CE) was used to determine the whey protein to total protein ratio in raw and UHT milk samples with different degrees of proteolysis causedbystorage. Inrawmilks,theanalysis ofsamples taken at regular times demonstrated the influence of proteolysis in the whey protein to total protein determi- nation, which was overestimated after 4 d of storage. In UHT milks, the overestimation of

  5. Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide

    PubMed Central

    Birdsall, Robert E.; Koshel, Brooke M.; Hua, Yimin; Ratnayaka, Saliya N.; Wirth, Mary J.

    2013-01-01

    Sieving of proteins in silica colloidal crystals of mm dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. The model also fits the data for electrophoresis in a highly ordered colloidal crystal formed in a channel, where the unknown pore radius was used as a fitting parameter. Plate heights as small as 0.4 ?m point to the potential for miniaturized separations. Band broadening increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. The results quantitatively support the notion that sieving occurs for proteins in silica colloidal crystals, and facilitate design of new separations that would benefit from miniaturization. PMID:23229163

  6. SEPARATION OF GLUTEN PROTEINS BY HIGH PERFORMANCE CAPILLARY ELECTROPHORESIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High performance capillary electrophoresis (HPCE) is an analytical method that uses a voltage differential to accurately move solvents and solutes through a capillary. HPCE is a relative newcomer to the field of cereal chemistry, it utilizes small inner diameter capillaries as an anti-convective med...

  7. A simple cellulose acetate membrane-based small lanes technique for protein electrophoresis.

    PubMed

    Na, Na; Liu, Tingting; Yang, Xiaojun; Sun, Binjie; Ouyang, Jenny; Ouyang, Jin

    2012-08-01

    Combining electrophoresis with a cellulose acetate membrane-based technique, we developed a simple and low-cost method, named cellulose acetate membrane-based small lanes (CASL), for protein electrophoresis. A home-made capillary plotter controlled by a 3D moving stage was used to create milli-to-micro channels by printing poly(dimethylsiloxane) on to a hydrophilic cellulose acetate membrane. In the hydrophilic channels, 5 nL protein mixture was separated on the basis of electro-migration under an electric field. Compared with polyacrylamide gel electrophoresis (PAGE), CASL resulted in higher protein signal intensity for separation of mixtures containing the same mass of protein. The platform was easily fabricated at low cost (approx. $0.005 for each 1-mm-wide channel), and separation of three protein mixtures was completed in 15 min. Both electrophoresis time and potential affected the separation. Rather than chromatographic separation, this method accomplished application of microchannel techniques for cellulose acetate membrane-based protein electrophoresis. It has potential in proteomic analysis, especially for rapid, low-cost, and low-volume sample analysis in clinical diagnosis. PMID:22752445

  8. Viral quantitative capillary electrophoresis for counting and quality control of RNA viruses.

    PubMed

    Azizi, Afnan; Mironov, Gleb G; Muharemagic, Darija; Wehbe, Mohamed; Bell, John C; Berezovski, Maxim V

    2012-11-01

    The world of health care has witnessed an explosive boost to its capacity within the past few decades due to the introduction of viral therapeutics to its medicinal arsenal. As a result, a need for new methods of viral quantification has arisen to accommodate this rapid advancement in virology and associated requirements for efficiency, speed, and quality control. In this work, we apply viral quantitative capillary electrophoresis (viral qCE) to determine (i) the number of intact virus particles (ivp) in viral samples, (ii) the amount of DNA contamination, and (iii) the degree of viral degradation after sonication, vortexing, and freeze-thaw cycles. This quantification method is demonstrated on an RNA-based vesicular stomatitis virus (VSV) with oncolytic properties. A virus sample contains intact VSV particles as well as residual DNA from host cells, which is regulated by WHO guidelines, and may include some carried-over RNA. We use capillary zone electrophoresis with laser-induced fluorescent detection to separate intact virus particles from DNA and RNA impurities. YOYO-1 dye is used to stain all DNA and RNA in the sample. After soft lysis of VSV with proteinase K digestion of viral capsid and ribonucleoproteins, viral RNA is released. Therefore, the initial concentration of intact virus is calculated based on the gain of a nucleic acid peak and an RNA calibration curve. After additional NaOH treatment of the virus sample, RNA is hydrolyzed leaving residual DNA only, which is also calculated by a DNA calibration curve made by the same CE instrument. Viral qCE works in a wide dynamic range of virus concentrations from 10(8) to 10(13) ivp/mL. It can be completed in a few hours and requires minimum optimization of CE separation. PMID:23046075

  9. A quantitative method for measuring protein phosphorylation

    Microsoft Academic Search

    J. Andres Mckenzie; Phyllis R. Strauss

    2003-01-01

    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).

  10. Recovery of Native Proteins from Preparative Electrophoresis Gel Slices by Reverse Polarity ElutioN

    Microsoft Academic Search

    Aaron S. Abramovitz; Verrell Randolph; Aruna Mehra; Sylvia Chnstakos

    1984-01-01

    A technique for high yield recovery of native, biologically active proteins from preparative polyacrylamide gel slices by reverse polarity elution is described. No apparatus other than the standard slab gel electrophoresis system is required. Several proteins have been recovered in biologically active form at a 90% yield, in quantities ranging from 0.4 mg to 4.2 mg. The method is effective

  11. Detergents and chaotropes for protein solubilization before two-dimensional electrophoresis Thierry RABILLOUD*,

    E-print Network

    Paris-Sud XI, Université de

    Detergents and chaotropes for protein solubilization before two-dimensional electrophoresis Thierry mainly with chaotropes and new detergents, which are both able to enhance protein solubility. The input. Chaotropes. Detergents. Zwitterionic detergents. hal-00373287,version1-3Apr2009 Author manuscript, published

  12. Image processing methods and algorithms for accurate protein spot detection in 2-dimensional gel electrophoresis (2DGE)

    E-print Network

    Kouroupetroglou, Georgios

    -dimensional gel electrophoresis, denoising, image segmentation, spot detection, spot quantification, spot modeling electrophoresis (2DGE) and gives rise to protein spots of irregular shapes and sizes on the gel. Once proteinsImage processing methods and algorithms for accurate protein spot detection in 2-dimensional gel

  13. Bence-Jones protein - quantitative

    MedlinePLUS

    Immunoglobulin light chains - urine; Urine Bence-Jones protein ... Bence-Jones proteins are a part of regular antibodies called light chains. These proteins are not normally in urine. Sometimes, when ...

  14. Quantitative determination of amino acids in functional foods by microchip electrophoresis.

    PubMed

    Ueno, Hiroko; Wang, Jun; Kaji, Noritada; Tokeshi, Manabu; Baba, Yoshinobu

    2008-03-01

    Microchip electrophoresis (MCE), a first-generation micrototal analysis system, has emerged during the miniaturization phase of food analysis. Based on the micellar electrokinetic chromatography mode, a simple and fast MCE method with light emitting diode-induced fluorescence detection was developed for quantitative analysis of amino acids in three different kinds of functional foods, viz. sports beverages, jelly-form beverages, and tablet-form functional foods. In contrast to the glass microchip, we improved the separation of amino acids on a poly(methyl methacrylate) (PMMA) chip by addition of cationic starch derivatives. 4-fluoro-7-nitro-2,1,3-benzoxadiazole, which has a short labeling time for amino acids, was used as the fluorescently labeled dye. This MCE method takes less than 10 min of total analysis time including sample preparation and analysis of amino acids in functional foods on a PMMA chip. The results show that this approach has the potential to be a fast and simple method for amino acid analysis in functional foods. PMID:18266297

  15. Quantitative determination of serum iron in human blood by high-performance capillary electrophoresis

    Microsoft Academic Search

    Ping Che; Jie Xu; Honglan Shi; Yinfa Ma

    1995-01-01

    A capillary electrophoretic (HPCE) method that can be used to quantitatively determine trace amounts of iron has been developed and applied to determine the iron level in human serum. After precipitation of serum proteins, Fe(III) in the serum is reduced to Fe(II) with hydroxylamine hydrochloride, and a stable Fe(II)-1,10-phenanthroline complex is formed by adding 1,10-phenanthroline to the supernatant containing 2.5

  16. Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein-Protein Interactions

    PubMed Central

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

    2013-01-01

    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 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 3,443 compounds and results compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that reconfirmed in subsequent testing suggesting greater specificity. This finding was attributed to 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

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

    PubMed

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

    2013-10-15

    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

  18. Protein electrophoresis as a diagnostic and prognostic tool in raptor medicine.

    PubMed

    Tatum, L M; Zaias, J; Mealey, B K; Cray, C; Bossart, G D

    2000-12-01

    Plasma proteins of 139 healthy adult birds of prey from 10 species were separated by electrophoresis to characterize and document normal reference ranges and species-specific electrophoretic patternsand to evaluate the value of this technique for health screening, disease diagnosis, and prognostic indication. Species studied included bald eagle (Haliaeetus leucocephalus), red-tailed hawk (Buteo jamaicensis), barn owl (Tyto alba), great horned owl (Bubo virginianus), turkey vulture (Cathartes aura), Harris' hawk (Parabuteo unicinctus), Stellar's sea eagle (Haliaeetus pelagicus), barred owl (Strix varia), screech owl (Otus asio), and black vulture (Coragyps atratus). Several clinical cases show the diagnostic/therapeutic value of protein electrophoresis in raptors. This study establishes species-specific reference ranges for several birds of prey and discusses the benefit of electrophoresis as a diagnostic technique in health screens, as a diagnostic aid in conjunction with other tests, and as a prognostic indicator in clinical evaluation of raptors. PMID:11428396

  19. Determination of free L- and D-alanine in hydrolysed protein fertilisers by capillary electrophoresis.

    PubMed

    Cavani, Luciano; Ciavatta, Claudio; Gessa, Carlo

    2003-01-24

    of racemisation of hydrolysed protein fertilisers (HPFs) using an The objective of this study was to determine the degree inexpensive and easy to handle analytical method for qualitative control of the products. Using a polyacrylamide coated capillary and a run buffer containing 0.1 M Tris-borate+2.5 mM EDTA-Na2+0.1% sodium dodecylsulfate+10 mM beta-cyclodextrin a quantitative separation of D- and L-alanine (Ala) was made from an not treated HPF sample derivatised with dansyl chlorine by capillary electrophoresis. The D-Ala:[D-Ala+L-Ala] ratio, called degree of racemisation (RD), was calculated. The analysis of ten commercial HPFs has shown that more than 60% of HPFs have an RD > or = 40%. while only one product has shown an RD <5%. These results showed that most of the HPFs on the market are obtained with strong hydrolytic processes and high contents of D-amino acids are probably less effective as plant nutrients or even potentially dangerous to plants. PMID:12580515

  20. [Total protein analysis by two-dimensional electrophoresis in cysticerci of Taenia solium and Taenia asiatica].

    PubMed

    Fang, Wen; Xiao, Liang-Liang; Bao, Huai-En; Mu, Rong

    2011-06-01

    Two 20-day-old three-way crossed hybrid pigs were infected with 80000 Taenia solium or T. asiatica eggs, respectively. Immature cysticerci of the two species in liver were collected at 40 days after infection. The total proteins were separated by two-dimensional electrophoresis, and differentially expressed proteins were analyzed by Image-Master 2D Platinum 6.0 software. The results showed that there were (236 +/- 12) and (231 +/- 14) protein spots in 2D electrophoresis gel images of T. solium and T. asiatica, respectively, with 3 proteins up-regulated and 7 proteins down-regulated in T. solium cysticercus by 2-fold or more compared with those in T. asiatica cysticercus. PMID:21970107

  1. Detection of ? and ? Light Chain Monoclonal Proteins in Human Serum: Automated Immunoassay versus Immunofixation Electrophoresis

    PubMed Central

    Jaskowski, Troy D.; Litwin, Christine M.; Hill, Harry R.

    2006-01-01

    Recently, turbidimetric immunoassays for detecting and quantifying ? and ? free light chains (FLC) have become available and are promoted as being more sensitive than immunofixation electrophoresis (IFE) in detecting FLC monoclonal proteins. In this study, we assessed the ability of these turbidimetric assays to detect serum monoclonal proteins involving both free and heavy-chain-bound ? and ? light chains compared to standard immunofixation electrophoresis. Sera demonstrating a restricted band of protein migration (other than a definite M spike) by serum protein electrophoresis (SPE), which may represent early monoclonal proteins, were also examined. When compared to IFE, percent agreement, sensitivity, and specificity for the ?-FLC and ?-FLC were 94.6, 72.9, and 99.5% and 98.5, 91.4, and 99.7%, respectively, in detecting monoclonal proteins involving free and heavy-chain-bound light chains. The majority of sera (73.7%) demonstrating a restricted band of protein migration on SPE demonstrated abnormal IFE patterns suggestive of multiple myeloma or monoclonal gammopathy of unknown significance, but gave normal ?/? FLC ratios using the turbidimetric immunoassays. In conclusion, the ? and ? FLC assays are significantly less sensitive (72.9 to 91.4%) than IFE, but specific in detecting serum monoclonal proteins. Moreover, the ?/? ratio has little value in routine screening since the majority of sera with abnormal IFE patterns had normal ?/? FLC ratios. PMID:16467338

  2. Journal of Chromatography B, 839 (2006) 112117 Capillary electrophoresis of peptides and proteins

    E-print Network

    Miksik, Ivan

    Journal of Chromatography B, 839 (2006) 112­117 Capillary electrophoresis of peptides and proteins of two separation techniques is important in the resolution of complex This paper was presented at the 4 of the sample. There is one disad- vantage in ACE compare to affinity chromatography--affinity ligand

  3. SEPARATION AND IDENTIFICATION OF SOYBEAN LEAF PROTEINS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS AND MASS SPECTROMETRY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted las...

  4. Sequence analyses of rat liver cytosolic proteins separated by gel electrophoresis using MALDI mass spectrometry

    E-print Network

    Ens, Werner

    was performed by in-gel tryptic digestion, MALDI mass spectrometry, and database search with peptide mass method by MALDI mass spectrometry also revealed that chemical modifications occurred at the residuesSequence analyses of rat liver cytosolic proteins separated by gel electrophoresis using MALDI mass

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

    ERIC Educational Resources Information Center

    Kim, Thomas D.; Craig, Paul A.

    2010-01-01

    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…

  6. Bargain Electrophoresis.

    ERIC Educational Resources Information Center

    Maderia, Vitor M. C.; Pires, Euclides M. V.

    1986-01-01

    Discusses the value of electrophoresis in the fields of protein chemistry and biochemistry. Describes how to build an inexpensive electrophoresis setup for use in either research or teaching activities. Details the construction of both the separating device and the power supply. (TW)

  7. Pneumatic microvalve-based hydrodynamic sample injection for high-throughput, quantitative zone electrophoresis in capillaries.

    PubMed

    Kelly, Ryan T; Wang, Chenchen; Rausch, Sarah J; Lee, Cheng S; Tang, Keqi

    2014-07-01

    A hybrid microchip/capillary electrophoresis (CE) system was developed to allow unbiased and lossless sample loading and high-throughput repeated injections. This new hybrid CE system consists of a poly(dimethylsiloxane) (PDMS) microchip sample injector featuring a pneumatic microvalve that separates a sample introduction channel from a short sample loading channel, and a fused-silica capillary separation column that connects seamlessly to the sample loading channel. The sample introduction channel is pressurized such that when the pneumatic microvalve opens briefly, a variable-volume sample plug is introduced into the loading channel. A high voltage for CE separation is continuously applied across the loading channel and the fused-silica capillary separation column. Analytes are rapidly separated in the fused-silica capillary, and following separation, high-sensitivity MS detection is accomplished via a sheathless CE/ESI-MS interface. The performance evaluation of the complete CE/ESI-MS platform demonstrated that reproducible sample injection with well controlled sample plug volumes could be achieved by using the PDMS microchip injector. The absence of band broadening from microchip to capillary indicated a minimum dead volume at the junction. The capabilities of the new CE/ESI-MS platform in performing high-throughput and quantitative sample analyses were demonstrated by the repeated sample injection without interrupting an ongoing separation and a linear dependence of the total analyte ion abundance on the sample plug volume using a mixture of peptide standards. The separation efficiency of the new platform was also evaluated systematically at different sample injection times, flow rates, and CE separation voltages. PMID:24865952

  8. Comparison of proteins from Mycobacterium fortuitum, Mycobacterium nonchromogenicum and Mycobacterium terrae using flat bed electrophoresis.

    PubMed

    Vanden Berghe, D A; Pattyn, S R

    1979-04-01

    Polyacrylamide gel electrophoresis of bacterial lysates in a flat bed gives a linear relationship between 1n mol. wt of the proteins and the square root of their migration distances, thereby allowing standardization of different electrophoresis runs and precise comparison between homologous bands. The results obtained with Mycobacterium fortuitum, M. terrae and M. nonchromogenicum strains were used in numerical analysis. Mycobacterium fortuitum and M. nonchromogenicum showed a greater internal similarity than M. terrae, while two strains of the latter clustered with M. nonchromogenicum. The method described allows the comparison of mycobacteria with different generation times and provides a large number of good characters for numerical taxonomy. PMID:479829

  9. Use of DNA Ladders for Reproducible Protein Fractionation by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

    E-print Network

    Chait, Brian T.

    . Keywords: DNA · quantitation · fractionation · SILAC · mass spectrometry · proteomics Introduction In shotgun proteomics, protein digests are usually analyzed by liquid chromatography-tandem mass spectrometry with mass spectrometry. To evaluate the reproducibility of DNA-ladder-assisted gel cutting for quantitative

  10. The quantification of oxygen toxicity by the technique of cellulose acetate electrophoresis of rat serum proteins

    E-print Network

    Barker, Marcia Wagner

    1979-01-01

    by cardiac puncture before and after exposure periods and serum was aspirated from the clotted and centrifuged samples. Serum samples were subjected to electrophoresis on cellulose acetate and examined for qualitative and quantitative changes... be of importance in oxygen toxicity studies and in the treatment of newborns with hyaline membrane disease. Although the process of hyalinization is not well understood, work done by Phillips, Wyte, Weeks and Soloway (116) and by Fujikura (63) indicated a...

  11. Identification of methanococcus jannaschii proteins in 2-D gel electrophoresis patterns by mass spectrometry.

    SciTech Connect

    Liang, X.

    1998-06-10

    The genome of Methanococcus jannaschii has been sequenced completely and has been found to contain approximately 1,770 predicted protein-coding regions. When these coding regions are expressed and how their expression is regulated, however, remain open questions. In this work, mass spectrometry was combined with two-dimensional gel electrophoresis to identify which proteins the genes produce under different growth conditions, and thus investigate the regulation of genes responsible for functions characteristic of this thermophilic representative of the methanogenic Archaea.

  12. Two-dimensional SDS-polyacrylamide gel electrophoresis of heat-modifiable outer-membrane proteins.

    PubMed

    Russell, R R

    1976-01-01

    An examination has been made of the effect which temperature of solubilization has upon the subsequent migration in SDS-polyacrylamide gel electrophoresis of proteins from the cell envelopes of Escherichia coli K12 and Neisseria sicca ATCC 9913. Conventional electrophoresis in tubes revealed substantial differences in the staining patterns of gels, depending upon whether the envelope samples were solubilized at 37 degrees C or 100 degrees C; in the case of N. sicca at least 6 of 13 discernible bands displayed heat-modifiable behavior. The relationship of the bands produced by each of the two temperatures was investigated by a two-dimensional electrophoresis procedure, in which a sample was solubilized at 37 degrees C and run in a usual cylindrical gel; the entire gel was then resolubilized at 100 degrees C, and laid along an acrylamide slab for electrophoresis in the second dimension. It was found that "free endotoxin" of both organisms examined contained the same major proteins as the total envelope fraction, and that these free endotoxin proteins showed the same heat-modifiable properties as when present in total envelopes. PMID:1252992

  13. Proteoliposome-based capillary electrophoresis for screening membrane protein inhibitors.

    PubMed

    Li, Bing; Lv, Xuefei; Geng, Lina; Qing, Hong; Deng, Yulin

    2012-08-01

    A method for screening of monoamine oxidase (MAO) inhibitor was carried out using capillary electrophoresis (CE) based on the interaction of MAO and its substrate kynuramine (Kyn). Bioactive proteoliposome was reconstituted by liposome and MAO and then was applied as the pseudostationary phase (PSP) of CE to mimic the interaction between the enzyme and its substrate. N-prolmrgyl-R-2-heptylamine (R-2-HPA) and rasagiline [N-propargyl-1-(R)-aminoindan], which are two kinds of MAO inhibitors, were added into the running buffers containing proteoliposome. The results showed that the relative migration time ratio (RMTR × 10(-1)) values of Kyn were enhanced from 8.88 to 9.31 with an increase of the concentrations of rasagiline from 10(-6) to 1 mM. However, the RMTR values of Kyn were enhanced from 8.83 to 9.14 with an increase of the concentrations of R-2-HPA from 10(-6) to 1 mM. The RMTR value of Kyn in the presence of rasagiline was larger than that in the presence of R-2-HPA when rasagiline and R-2-HPA were at the same concentration. The results indicated that the interaction between Kyn and MAO was weakened with the increase of the inhibitors. In addition, the results of offline incubation showed that the inhibitions of rasagiline were 100.0, 72.1, 51.8 and 5.4% at the concentration of 1, 10(-2), 10(-4) and 10(-6) mM; moreover, the inhibitions of R-2-HPA were 70.0, 44.9, 4.1 and 0.9% at the concentrations of 1, 10(-2), 10(-4) and 10(-6) mM. The inhibition efficiency of rasagiline was stronger than that of R-2-HPA at the same concentration. Additionally, the interaction between Kyn and liposome was also investigated. This newly developed method might provide a potential tool for screening MAO inhibitor. PMID:22547660

  14. Speciation of protein-bound trace elements by gel electrophoresis and atomic spectrometry.

    PubMed

    Ma, Renli; McLeod, Cameron W; Tomlinson, Kerry; Poole, Robert K

    2004-08-01

    The metabolism of trace elements, in particular their binding to proteins in biological systems is of great importance in biochemical, toxicological, and pharmacological studies. As a result there has been a sustained interest over the last two decades in the speciation of protein-bound metals. Various analytical approaches have been employed, combining efficient separation of metalloproteins by liquid chromatography or electrophoresis with high-sensitivity elemental detection. Slab-gel electrophoresis (GE) is a key platform for high-resolution protein separation, and has been combined with autoradiography and various atomic spectrometric techniques for in-gel determination of protein-bound metals. Recently, the combination of GE with state-of-the-art inductively coupled plasma-mass spectrometry (ICP-MS), particularly when linked to laser ablation (LA) for direct gel interrogation, has opened up new opportunities for rapid characterization of metalloproteins. The use of GE and atomic spectrometry for the speciation of protein-bound trace elements is reviewed in this paper. Technical requirements for gel electrophoresis/atomic spectrometric measurement are considered in terms of method compatibilities, detection capability and potential usefulness. The literature is also surveyed to illustrate current status and future trends. PMID:15300764

  15. Isolation of Spiroplasma citri membranes and characterization of membrane proteins by two-dimensional gel electrophoresis

    Microsoft Academic Search

    Philippe Simoneau; Jacques Labarère

    1988-01-01

    This paper describes a method for isolating plasma membranes fromSpiroplasma citri and for comparing membrane and cytoplasmic proteins by two-dimensional gel electrophoresis. Plasma membranes ofS. citri were stabilized against fragmentation by coating cell with Concanavalin A just prior to lysis. After lysis of the cells by ultrasonic irradiation, membranes were purified by differential centrifugation and step gradients. The purified fraction,

  16. Amino Acid Composition, Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L.) Proteins and Protein Fractionations

    PubMed Central

    Mao, Xiaoying; Hua, Yufei; Chen, Guogang

    2014-01-01

    As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE) showed that the isoelectric point was mainly in the range of 4.8–6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa. PMID:24473146

  17. Capillary zone electrophoresis for separation and quantitative determination of mexiletine and its main phase I metabolites.

    PubMed

    Bruno, Claudio; Cavalluzzi, Maria Maddalena; Carocci, Alessia; Catalano, Alessia; Franchini, Carlo; Lentini, Giovanni

    2013-03-01

    The simultaneous separation and quantification of the analytes within the minimum analysis time and the maximum resolution and efficiency are the main objectives in the development of a capillary electrophoretic method for the determination of solutes. In this paper we describe a specific, sensitive and robust method, using capillary zone electrophoresis with internal standard and UV detection, for the separation and quantification of the anti-arrhythmic drug mexiletine, its main phase I metabolites, and its main nitrogenous degradation product. PMID:23826880

  18. Identification of protein binding sites in genomic DNA by two-dimensional gel electrophoresis.

    PubMed Central

    Boffini, A; Prentki, P

    1991-01-01

    We describe a simple two-dimensional electrophoresis procedure to identify the recognition sites of DNA-binding proteins within large DNA molecules. Using this approach, we have mapped E. coli IHF (Integration Host Factor) binding sites within phage Lambda (48 kb) and phage Mu (39 kb) DNA. We are also able to visualize IHF binding sites in E. coli chromosomal DNA (4,700 kb). We present an extension of this technique using direct amplification by PCR of the isolated restriction fragments, which should permit the cloning of a collection of recognition sequences for DNA binding proteins in complex genomes. Images PMID:1827523

  19. Red wine proteins: two dimensional (2-D) electrophoresis and mass spectrometry analysis.

    PubMed

    Mainente, Federica; Zoccatelli, Gianni; Lorenzini, Marilinda; Cecconi, Daniela; Vincenzi, Simone; Rizzi, Corrado; Simonato, Barbara

    2014-12-01

    The aim of the present study was to optimize protein extraction from red wine (cv. Cabernet) in order to obtain a separation by two-dimensional electrophoresis (2-DE) compatible with mass spectrometry identification. Proteins were denatured by sodium dodecyl-sulphate (SDS) and precipitated as potassium salts. The potassium-DS (KDS) protein complexes obtained were treated with different solutions in order to remove the detergent. Proteins were solubilized with different buffers and separated by different electrophoretic approaches [native, urea, acid urea PAGEs and isoelectric focusing (IEF)] as the first-dimension (1-DE). The best 2D separation was achieved by using 10% saccharose in the DS removal step, and 6-cyclohexylhexyl ?-d-maltoside detergent in the solubilisation buffer combined with the IEF approach. Several well focalized protein spots were obtained and analyzed through mass-spectrometry. PMID:24996352

  20. Optimisation of a simple and reliable label-free methodology for the relative quantitation of raw pork meat proteins.

    PubMed

    Gallego, Marta; Mora, Leticia; Aristoy, M Concepción; Toldrá, Fidel

    2015-09-01

    Recent advances in proteomics have become an indispensable tool for a fast, precise and sensitive analysis of proteins in complex biological samples at both, qualitative and quantitative level. In this study, a label-free quantitative proteomic methodology has been optimised for the relative quantitation of proteins extracted from raw pork meat. So, after the separation of proteins by one-dimensional gel electrophoresis and trypsin digestion, their identification and quantitation have been done using nanoliquid chromatography coupled to a quadrupole/time-of-flight (Q/ToF) mass spectrometer. Relative quantitation has been based on the measurement of mass spectral peak intensities, which have been described that are correlated with protein abundances. The results obtained regarding linearity, robustness, repeatability and accuracy show that this procedure could be used as a fast, simple, and reliable method to quantify changes in protein abundance in meat samples. PMID:25842311

  1. Reference intervals for acute phase protein and serum protein electrophoresis values in captive Asian elephants (Elephas maximus).

    PubMed

    Isaza, Ramiro; Wiedner, Ellen; Hiser, Sarah; Cray, Carolyn

    2014-09-01

    Acute phase protein (APP) immunoassays and serum protein electrophoresis (SPEP) are assays for evaluating the inflammatory response and have use as diagnostic tools in a variety of species. Acute phase proteins are markers of inflammation that are highly conserved across different species while SPEP separates and quantifies serum protein fractions based on their physical properties. In the current study, serum samples from 35 clinically healthy Asian elephants (Elephas maximus) were analyzed using automated assays for C-reactive protein, serum amyloid A, and haptoglobin and SPEP. Robust methods were used to generate reference intervals for the APPs: C-reactive protein (1.3-12.8 mg/l), serum amyloid A (0-47.5 mg/l), and haptoglobin (0-1.10 mg/ml). In addition, SPEP was performed on these samples to establish reference intervals for each protein fraction. A combination of APPs and SPEP measurements are valuable adjunctive diagnostic tools in elephant health care. PMID:25057161

  2. Effectiveness and limitation of two-dimensional gel electrophoresis in bacterial membrane protein proteomics and perspectives.

    PubMed

    Bunai, Keigo; Yamane, Kunio

    2005-02-01

    Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) using isoelectric focusing and SDS-PAGE in the first and second dimensions, respectively, is an established means of simultaneously separating over 1000 proteins and two new types have recently been developed. These procedures have significant shortcomings such as low load ability and poor separation of hydrophobic, acidic and alkaline proteins. We therefore modified the protocols to analyze the Bacillus subtilis membrane proteome. The 2D-PAGE techniques effectively separated membrane proteins having one and two transmembrane segments but not those with more than four. Compared with new LC/MS/MS procedures that are independent of electrophoretic separation, 2D-PAGE can globally analyze and quantify proteins at various stages of the cell cycle when labeled with isotopes such as 35S-methionine or the stable isotope, 15N. PMID:15652812

  3. Quantitative study of protein-protein interactions by quartz nanopipettes.

    PubMed

    Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

    2014-09-01

    In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions. PMID:25060094

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

    PubMed Central

    Gauci, Victoria J.; Wright, Elise P.

    2010-01-01

    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. PMID:21686332

  5. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    DOEpatents

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

    1989-01-01

    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.

  6. Polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins of cutaneous Propionibacterium species.

    PubMed

    Nordstrom, K M

    1985-02-01

    Polyacrylamide gel electrophoresis (PAGE) was applied to the study of whole-cell proteins of cutaneous propionibacteria in an attempt to characterise possible protein patterns that may be typical for strains isolated from acne skin. Isolates were obtained from the faces of 33 individuals aged 7-16 years. Some of these subjects had apparently normal healthy skin, whereas others had acne vulgaris of varying severity. Twenty-five facial isolates of Propionibacterium acnes and eight of P. granulosum were studied. A further seven axillary strains of P. avidum were included for purely taxonomic interest. No particular protein pattern was characteristic of an isolate from acne skin; in fact the P. acnes strains from all sources appeared to be identical. PMID:3155801

  7. Global approaches to quantitative analysis of gene-expression patterns observed by use of two-dimensional gel electrophoresis

    SciTech Connect

    Anderson, N.L.; Hofmann, J.P.; Gemmell, A.; Taylor, J.

    1984-01-01

    A major difficulty in the use of two-dimensional protein maps to identify and classify cell types is the problem of acquiring, selecting, and analyzing quantitative data on hundreds of protein spots. Here the authors use methods of multivariate statistics to analyze the differences among a panel of human cell lines, in some cases involving quantitative data on more than 250 proteins. Principal-component and cluster-analysis techniques show that the lines can be easily distinguished, even by using the subset of proteins present in all cells. A preliminary analysis of the protein changes brought about by phorbol ester-induced differentiation of the line U937 is included. 18 references, 6 figures.

  8. Application of the commercial gel electrophoresis apparatus with intermittent fluorescence scanning to a nonfluorescing protein.

    PubMed

    Chen, N; Chrambach, A

    1996-11-01

    Gel electrophoretic instrumentation has taken a quantum jump forward with the commercial introduction of an apparatus which, after loading of the sample and initiation of electrophoresis, provides real-time gel patterns at desired time intervals, with a computer printout of mobility values characterizing each band and the means to isolate each desired band with known and maximizeable recovery. However, a major limitation of that apparatus has been that it employs fluorescence detection and therefore requires the fluorescent labeling of the macromolecules of interest. That limitation was first overcome by E. Gombocz and E. Cortez (Application Note 8, 1994, LabIntelligence, Belmont, CA) in the detection of nonfluorescing carrier ampholytes. In that application, fluorescent, immobile (uncharged) umbelliferone was added to the gel to provide a uniform background of fluorescence upon excitation at 280-360 nm. The isoelectric carrier ampholyte zones could be detected as inverted peaks due to their reduction of the fluorescence intensity of umbelliferone. A similar approach was applied to a representative SDS-protein, conalbumin-SDS, in the present study, replacing umbelliferone in the gel by a fluorescing paper sheet in contact with the lower external surface of the electrophoresis cell. Passage of the proteins reduced the intensity of the light excitation incident on the fluorescent paper so as to decrease the emitted fluorescence signal and allow for the detection of the proteins as "inverted peaks." Presumably, the reduction of background fluorescence is due to the absorbance at 280 nm of the protein passing through the gel, and reduction of the incident light intensity by that absorbance. The resulting detection of the representative unlabeled SDS-protein by "fluorescence reduction" was found to be less sensitive by a factor of 10-20 than detection of the fluorescently labeled protein (at a molar ratio of fluorescein carboxylate to conalbumin of 1/1). The area of the inverted bands of conalbumin-SDS was found to be independent of migration distance. PMID:8923965

  9. Highly sensitive capillary electrophoresis-mass spectrometry for rapid screening and accurate quantitation of drugs of abuse in urine.

    PubMed

    Kohler, Isabelle; Schappler, Julie; Rudaz, Serge

    2013-05-30

    The combination of capillary electrophoresis (CE) and mass spectrometry (MS) is particularly well adapted to bioanalysis due to its high separation efficiency, selectivity, and sensitivity; its short analytical time; and its low solvent and sample consumption. For clinical and forensic toxicology, a two-step analysis is usually performed: first, a screening step for compound identification, and second, confirmation and/or accurate quantitation in cases of presumed positive results. In this study, a fast and sensitive CE-MS workflow was developed for the screening and quantitation of drugs of abuse in urine samples. A CE with a time-of-flight MS (CE-TOF/MS) screening method was developed using a simple urine dilution and on-line sample preconcentration with pH-mediated stacking. The sample stacking allowed for a high loading capacity (20.5% of the capillary length), leading to limits of detection as low as 2 ng mL(-1) for drugs of abuse. Compound quantitation of positive samples was performed by CE-MS/MS with a triple quadrupole MS equipped with an adapted triple-tube sprayer and an electrospray ionization (ESI) source. The CE-ESI-MS/MS method was validated for two model compounds, cocaine (COC) and methadone (MTD), according to the Guidance of the Food and Drug Administration. The quantitative performance was evaluated for selectivity, response function, the lower limit of quantitation, trueness, precision, and accuracy. COC and MTD detection in urine samples was determined to be accurate over the range of 10-1000 ng mL(-1) and 21-1000 ng mL(-1), respectively. PMID:23680557

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

    PubMed

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

    1989-06-01

    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. PMID:2722241

  11. Quantitative biophysical characterization of intrinsically disordered proteins.

    PubMed

    Gibbs, Eric B; Showalter, Scott A

    2015-02-17

    Intrinsically disordered proteins (IDPs) are broadly defined as protein regions that do not cooperatively fold into a spatially or temporally stable structure. Recent research strongly supports the hypothesis that a conserved functional role for structural disorder renders IDPs uniquely capable of functioning in biological processes such as cellular signaling and transcription. Recently, the frequency of application of rigorous mechanistic biochemistry and quantitative biophysics to disordered systems has increased dramatically. For example, the launch of the Protein Ensemble Database (pE-DB) demonstrates that the potential now exists to refine models for the native state structure of IDPs using experimental data. However, rigorous assessment of which observables place the strongest and least biased constraints on those ensembles is now needed. Most importantly, the past few years have seen strong growth in the number of biochemical and biophysical studies attempting to connect structural disorder with function. From the perspective of equilibrium thermodynamics, there is a clear need to assess the relative significance of hydrophobic versus electrostatic forces in IDP interactions, if it is possible to generalize at all. Finally, kinetic mechanisms that invoke conformational selection and/or induced fit are often used to characterize coupled IDP folding and binding, although application of these models is typically built upon thermodynamic observations. Recently, the reaction rates and kinetic mechanisms of more intrinsically disordered systems have been tested through rigorous kinetic experiments. Motivated by these exciting advances, here we provide a review and prospectus for the quantitative study of IDP structure, thermodynamics, and kinetics. PMID:25631161

  12. Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients.

    E-print Network

    Paris-Sud XI, Université de

    pH gradients. Thierry Rabilloud 1*, Céline Adessi 2, Anne Giraudel 3 and Joël Lunardi 1 1: CEA resolution two-dimensional electrophoresis. IEF with immobilized pH gradients leads to severe quantitative) aminomethane; %C: ratio (in percent) of crosslinker to total monomers; CA-IEF: isoelectric focusing in pH

  13. Precise, fast, and flexible determination of protein interactions by affinity capillary electrophoresis: part 3: anions.

    PubMed

    Xu, Yuanhong; Redweik, Sabine; El-Hady, Deia Abd; Albishri, Hassan M; Preu, Lutz; Wätzig, Hermann

    2014-08-01

    The binding of physiologically anionic species or negatively charged drug molecules to proteins is of great importance in biochemistry and medicine. Since affinity capillary electrophoresis (ACE) has already proven to be a suitable analytical tool to study the influence of ions on proteins, this technique was applied here for comprehensively studying the influence of various anions on proteins of BSA, ?-lactoglobulin, ovalbumin, myoglobin, and lysozyme. The analysis was performed using different selected anions of succinate, glutamate, phosphate, acetate, nitrate, iodide, thiocyanate, and pharmaceuticals (salicylic acid, aspirin, and ibuprofen) that exist in the anionic form at physiological pH 7.4. Due to the excellent repeatability and precision of the ACE measurements, not necessarily strong but significant influences of the anions on the proteins were found in many cases. Different influences in the observed bindings indicated change of charge, mass, or conformational changes of the proteins due to the binding with the studied anions. Combining the mobility-shift and pre-equilibrium ACE modes, rapidity and reversibility of the protein-anion bindings were discussed. Further, circular dichroism has been used as an orthogonal approach to characterize the interactions between the studied proteins and anions to confirm the ACE results. Since phosphate and various anions from amino acids and small organic acids such as succinate or acetate are present in very high concentrations in the cellular environment, even weak influences are certainly relevant as well. PMID:24436007

  14. Capillary affinity electrophoresis for the screening of post-translational modification of proteins with carbohydrates.

    PubMed

    Nakajima, Kazuki; Oda, Yasuo; Kinoshita, Mitsuhiro; Kakehi, Kazuaki

    2003-01-01

    Glycosylation is one of the most important post-translational events for proteins, affecting their functions in health and disease, and plays significant roles in various information traffics for intracellular and intercellular biological events (Hancock, W. S. J. Proteome Res. 2002, 1, 297). We have attempted to obtain the information on the numbers and amounts of carbohydrate chains. Interaction between carbohydrate chains and proteins that recognize them is a target to understand the biological roles of glycosylation. To date, there have been a few strategies for simultaneous analysis of the interactions between complex mixtures of carbohydrates and proteins. Here, we report an approach to categorize carbohydrate chains using a few glycoprotein samples as models for the studies on the analysis of post-translational modification of proteins with carbohydrates. A combination of some specific lectins was used as carbohydrate-binding proteins. The method is based on high-resolution separation of fluorescent-labeled carbohydrates by capillary electrophoresis with laser-induced fluorescent detection in the presence of carbohydrate-binding proteins at different concentrations. The present technique affords (1) simultaneous determination of carbohydrate chains, (2) binding specificity of the constituent carbohydrate chains to specific proteins, and (3) kinetic data such as the association constant of each carbohydrate. We found that the lectins employed in the present study could discriminate subtle difference in linkages and resolved the carbohydrate mixtures. The results will be useful, for example, to understand the biological events expressed with carbohydrate changes on the cell surface. PMID:12643546

  15. Urine proteins identified by two-dimensional differential gel electrophoresis facilitate the differential diagnoses of scrapie.

    PubMed

    Lamoureux, Lise; Simon, Sharon L R; Plews, Margot; Ruddat, Viola; Brunet, Simone; Graham, Catherine; Czub, Stefanie; Knox, J David

    2013-01-01

    The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry. PMID:23704971

  16. Urine Proteins Identified by Two-Dimensional Differential Gel Electrophoresis Facilitate the Differential Diagnoses of Scrapie

    PubMed Central

    Lamoureux, Lise; Simon, Sharon L. R.; Plews, Margot; Ruddat, Viola; Brunet, Simone; Graham, Catherine; Czub, Stefanie; Knox, J. David

    2013-01-01

    The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry. PMID:23704971

  17. Comparison of protein expression profiles between three Perkinsus spp., protozoan parasites of molluscs, through 2D electrophoresis and mass spectrometry.

    PubMed

    Fernández-Boo, S; Chicano-Gálvez, E; Alhama, J; Barea, J L; Villalba, A; Cao, A

    2014-05-01

    The genus Perkinsus includes protozoan parasites of a wide range of marine molluscs worldwide, some of which have been responsible for heavy mollusc mortalities and dramatic economic losses. This study was performed with the aim of increasing the knowledge of Perkinsus spp. proteome. Proteins extracted from in vitro cultured cells of three species of this genus, P. marinus, P. olseni and P. chesapeaki, were analysed using 2D electrophoresis. Four gels from each species were produced. Qualitative and quantitative comparisons among gels were performed with Proteamweaver software. Cluster analysis grouped the four gels of each Perkinsus sp.; furthermore, P. marinus and P. olseni gels were grouped in a cluster different from P. chesapeaki. Around 2000 spots of each species were considered, from which 213 spots were common to the 3 species; P. chesapeaki and P. marinus shared 310 spots, P. chesapeaki and P. olseni shared 315 spots and P. marinus and P. olseni shared 242 spots. A number of spots were exclusive of each Perkinsus species: 1161 spots were exclusive of P. chesapeaki, 1124 of P. olseni and 895 of P. marinus. A total of 84 spots, including common and species-specific ones, were excised from the gels and analysed using MALDI-TOF and nESI-IT (MS/MS) techniques. Forty-two spots were successfully sequenced, from which 28 were annotated, most of them clustered into electron transport, oxidative stress and detoxification, protein synthesis, carbohydrate metabolism, signal transduction, metabolic process and proteolysis. PMID:24607654

  18. Quantitative study of protein-protein interactions by quartz nanopipettes

    NASA Astrophysics Data System (ADS)

    Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

    2014-08-01

    In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions. Electronic supplementary information (ESI) available: Determination of nanopipette diameter; surface modification scheme; numerical simulation; noise analysis; SPR experiments. See DOI: 10.1039/c4nr02964j

  19. Determination of the Sites of Posttranslational Modifications in the Charge Isomers of Bovine Myelin Basic Protein by Capillary Electrophoresis-Mass Spectroscopy

    E-print Network

    Zand, Robert

    Myelin Basic Protein by Capillary Electrophoresis-Mass Spectroscopy Robert Zand,* Michael X. Li, XiaoyingDa bovine myelin basic protein charge isomers C-1 to C-6 have been determined by the use of capillary, by a technique that combines capillary electrophoresis and mass spectroscopy. Bovine CNS myelin basic protein

  20. Quantitative Determination of Lercanidipine Enantiomers in Commercial Formulations by Capillary Electrophoresis

    PubMed Central

    Lourenço, Luciana Pereira; Aguiar, Fernando Armani; de Oliveira, Anderson Rodrigo Moraes; de Gaitani, Cristiane Masetto

    2015-01-01

    An enantioselective method based on capillary electrophoresis (CE) using cyclodextrin (CD) as chiral selector was developed and validated for determination of lercanidipine (LER) enantiomers, a drug calcium channel blocker which exerts antihypertensive effects of long duration, in a pharmaceutical formulation. Optimum separation of LER enantiomers was obtained on a 50?cm × 50??m id capillary using a sodium acetate buffer solution 200?mmol/L pH 4.0 containing 10?mmol/L of 2,3,6-o-methyl-?-cyclodextrin (TM-?-CD) as background electrolyte. The capillary temperature and voltage were 15°C and 25?kV, respectively, hydrodynamic injection and detection at 237?nm. Linearity was obtained in the range 12.5–100??g/mL for both enantiomers (r ? 0.995). The RSD (%) and relative errors (E, %) obtained in precision and accuracy studies (intraday and interday) were lower than 5%. After validation, the method was applied to quantify the enantiomers of LER in commercial tablets and the results were satisfactory in terms of accuracy and precision, both less than 5%. Therefore, this method was found to be appropriate for enantioselective quality control of LER enantiomers in pharmaceutical formulations.

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

    PubMed

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

    1999-08-01

    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

  2. Fluorescent monitoring of proteins during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting.

    PubMed

    Falk, B W; Elliott, C

    1985-02-01

    Fluorescent labeling of proteins was found to be a very sensitive and reliable alternative to conventional methods for monitoring proteins on Western blots. Proteins were labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) before SDS-PAGE. After electrophoresis and subsequent electro-blotting the fluorescent-labeled proteins were visible upon ultraviolet illumination of the nitrocellulose membranes, and could be photographed to yield an accurate record of the blots before subsequent serological analysis. The sensitivity for detecting MDPF-labeled proteins on nitrocellulose was 100-200 ng, 50 to 100 fold less sensitive than on gels. Fluorescent-labeled TMV and MStpV capsid proteins that were blotted onto nitrocellulose still reacted in serological tests and were detected when present in quantities as low as 100 pg. Fluorescent labeling allows accurate photographic records of the SDS-gel, blot and probed blot using only one sample, and no subsequent staining steps are required. PMID:3887982

  3. Serum protein electrophoresis by using high-resolution agarose gel in clinically healthy and Aspergillus species-infected falcons.

    PubMed

    Kummrow, Maya; Silvanose, Christudas; Di Somma, Antonio; Bailey, Thomas A; Vorbrüggen, Susanne

    2012-12-01

    Serum protein electrophoresis has gained importance in avian medicine during the past decade. Interpretation of electrophoretic patterns should be based on species-specific reference intervals and the electrophoresis gel system. In this study, serum protein electrophoresis by using high-resolution agarose gels was performed on blood samples collected from 105 falcons, including peregrine falcons (Falco peregrinus), gyrfalcons (Falco rusticolus), saker falcons (Falco cherrug), red-naped shaheens (Falco pelegrinoides babylonicus), and hybrid falcons, that were submitted to the Dubai Falcon Hospital (Dubai, United Arab Emirates) between 2003 and 2006. Reference values were established in clinically healthy birds and compared with values from falcons infected with Aspergillus species (n = 32). Falcons with confirmed aspergillosis showed significantly lower prealbumin values, which is a novel finding. Prealbumin has been documented in many avian species, but further investigation is required to illuminate the diagnostic significance of this negative acute-phase protein. PMID:23409432

  4. Quantitative capillary electrophoresis/ion spray tandem mass spectrometry determination of EDTA in human plasma and urine.

    PubMed

    Sheppard, R L; Henion, J

    1997-08-01

    A quantitative method has been developed for the determination of EDTA in human plasma and urine. The samples are prepared with automated anion-exchange solid-phase extraction using 100 microL of human plasma. The extracts are analyzed by capillary electrophoresis/mass spectrometry using selected reaction monitoring in the negative ion mode. Large-volume injections (10% of the CE capillary volume) are used to improve the concentration level of detection via field-amplified sample injection. The first reported validation of a CE/MS/MS technique was carried out for this method. Using a 13C stable-label isotope for the internal standard, the lower level of detection and lower level of quantitation were determined to be 7.3 and 14.6 ng/mL EDTA in human plasma, respectively. The injection precision had a relative standard deviation (RSD) of 6.1%. The intra-assay precision was less than 15% RSD. The intra-assay accuracy was less than +/- 12% bias from the nominal concentration. The interassay precision was less than 18% RSD and the interassay accuracy was less than +/- 9% bias from the nominal concentration. PMID:9253243

  5. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    DOEpatents

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

    1987-09-04

    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.

  6. Quantitative determination of phenolic compounds in lotus (Nelumbo nucifera) leaves by capillary zone electrophoresis.

    PubMed

    Do, Tho Chau Minh Vinh; Nguyen, Tuan Duc; Tran, Hung; Stuppner, Hermann; Ganzera, Markus

    2012-11-01

    The traditional use of lotus leaves as an anti-inflammatory remedy is associated with the occurrence of phenolic compounds. In this study the first CE method for the analysis of all major phenolic constituents in Nelumbo nucifera leaves is presented. It permits the separation of nine relevant markers in less than 10 min. The optimized procedure was fully validated and then used to analyze diverse samples collected in Vietnam. They revealed significant qualitative and quantitative differences depending on growing area and season. Yet, in all of them, quercetin-3-O-?-D-glucuronide, hyperoside, and isoquercitrin were the most dominant flavonoids. PMID:22923198

  7. Quantitative determination of (+)- and (-)-Gossypol in flower petals of selected cotton cultivars using capillary zone electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cottonseeds provide a high quality protein that is currently under utilized because of the presence of a toxic compound called gossypol. Gossypol is biosynthesized by the free radical coupling of two molecules of hemigossypol. This coupling reaction produces two optically active enantiomers. One ...

  8. DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.

    PubMed Central

    Xian, J; Harrington, M G; Davidson, E H

    1996-01-01

    A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method provides quantitative data in runs requiring < 20 min, from which dissociation constants are readily determined. As a test case we studied interactions of a developmentally important sea urchin embryo transcription factor, SpP3A2. As little as 2-10 x 10(6) molecules of specific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a single sea urchin egg lysed within the capillary column. Images Fig. 2 PMID:8552681

  9. Electrophoresis characterisation of protein as a method to establish the entomological origin of stingless bee honeys.

    PubMed

    Ramón-Sierra, Jesús Manuel; Ruiz-Ruiz, Jorge Carlos; de la Luz Ortiz-Vázquez, Elizabeth

    2015-09-15

    Increasing production of stingless-bee honey and the prospect of broader marker for natural and organic products indicate the need to establish parameters to determinate the entomological origin and authenticity of honey. In this research, honeys of Apis mellifera, Melipona beecheii and Trigona spp. were collected in Yucatan, Mexico. Stingless-bee honeys contained more water and less total sugars and reducing sugars. SDS-PAGE patterns show distinctive bands for each kind of honey. The SDS-PAGE pattern of A. mellifera proteins honey showed three bands with molecular weights between 10.2 and 74.8kDa, there were five proteins bands in M. beecheii honey with molecular weights between 6.1 and 97.0kDa and nine for Trigona spp. proteins between 9.3 and 86.7kDa. Conventional physicochemical parameters along with electrophoresis profiles of stingless-bee honeys proteins could be an alternative for determination of entomological origin. PMID:25863608

  10. Analysis of proteins expressed in rat plasma exposed to dioxin using 2-dimensional gel electrophoresis.

    PubMed

    Son, Won-Kyu; Lee, Do-Youn; Lee, Sung-Han; Joo, Won-A; Kim, Chan-Wha

    2003-12-01

    Dioxins are a class of polyhalogenated aromatic hydrocarbons that induces a wide spectrum of toxic responses in animals. In this study, two groups of Sprague-Dawley rats were exposed to 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD); one group received short-term exposure at a single dose of 1, 10, 20 or 50 microg/kg body weight and the other received long-term exposure to a daily low dose of 0.01, 0.1, 1 or 2.5 microg/kg body weight for one month. Two-dimensional electrophoresis was utilized to resolve the protein profile of rat plasma exposed to TCDD at different doses. One novel and three volume-increased spots were identified and characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and electrospray-ionization on quadropole-TOF2 mass spectrometry. The novel protein was identified as plasma glutathione peroxidase precursor and the volume-increased proteins were cytokeratin 8 polypeptide, Ig lambda-1 chain C region and Ig lambda-2 chain C region. These proteins may be used as biomarkers to diagnose dioxin exposure and may help in understanding the toxic effects of dioxins. PMID:14673789

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

    SciTech Connect

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

    1988-01-01

    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.

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

    SciTech Connect

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

    1980-07-01

    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.

  13. Analytical techniques for cell fractions. XXII. Two-dimensional analysis of serum and tissue proteins: multiple gradient-slab gel electrophoresis

    Microsoft Academic Search

    N. L. Anderson; N. G. Anderson

    1978-01-01

    Two-dimensional electrophoresis of proteins and protein subunits, employing isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate in the second, yields the highest resolutions currently available. In this paper separations in the second dimension are considered (the socalled DALT system). Methods for multiple-parallel casting of gradient gels in slab gel holders are described. The

  14. The use of seed protein electrophoresis in the study of phylogenetic relationships in Chili pepper ( Capsicum L.)

    Microsoft Academic Search

    R. C. Panda; O. Aniel Kumar; K. G. Raja Rao

    1986-01-01

    The seed protein profile of eight taxa of Chili peppers obtained by disc electrophoresis was found to be a diagnostic character in the study of phylogenetic relationships. The distinctness of each species and the wild and cultivated nature of concerned taxa has been confirmed. While the clustering of wildC. annuum var. ‘glabriusculum’ withC. baccatum types indicated that the former is

  15. Pneumatic Microvalve-Based Hydrodynamic Sample Injection for High-Throughput, Quantitative Zone Electrophoresis in Capillaries

    SciTech Connect

    Kelly, Ryan T.; Wang, Chenchen; Rausch, Sarah J.; Lee, Cheng S.; Tang, Keqi

    2014-07-01

    A hybrid microchip/capillary CE system was developed to allow unbiased and lossless sample loading and high throughput repeated injections. This new hybrid CE system consists of a polydimethylsiloxane (PDMS) microchip sample injector featuring a pneumatic microvalve that separates a sample introduction channel from a short sample loading channel and a fused silica capillary separation column that connects seamlessly to the sample loading channel. The sample introduction channel is pressurized such that when the pneumatic microvalve opens briefly, a variable-volume sample plug is introduced into the loading channel. A high voltage for CE separation is continuously applied across the loading channel and the fused silica capillary separation column. Analytes are rapidly separated in the fused silica capillary with high resolution. High sensitivity MS detection after CE separation is accomplished via a sheathless CE/ESI-MS interface. The performance evaluation of the complete CE/ESI-MS platform demonstrated that reproducible sample injection with well controlled sample plug volumes could be achieved by using the PDMS microchip injector. The absence of band broadening from microchip to capillary indicated a minimum dead volume at the junction. The capabilities of the new CE/ESI-MS platform in performing high throughput and quantitative sample analyses were demonstrated by the repeated sample injection without interrupting an ongoing separation and a good linear dependence of the total analyte ion abundance on the sample plug volume using a mixture of peptide standards. The separation efficiency of the new platform was also evaluated systematically at different sample injection times, flow rates and CE separation voltages.

  16. HIGH THROUGHPUT PROTEIN IDENTIFICATION USING 2-DIMENSIONAL DIFFERENCE GEL ELECTROPHORESIS AND ROBOTIC SPOT PICKING FOR ALUMINUM TOLERANCE-RELATED MAIZE ROOT TIP PROTEINS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two-dimensional difference gel electrophoresis (2-D DIGE) is the most effective method utilized to carry out gel-based quantitative proteomics. Unfortunately, the most popular image analysis software used to process DIGE images (DeCyder) produces picking coordinates in a format that is incompatible...

  17. A comprehensive platform to investigate protein-metal ion interactions by affinity capillary electrophoresis.

    PubMed

    Alhazmi, Hassan A; Nachbar, Markus; Albishri, Hassan M; El-Hady, Deia Abd; Redweik, Sabine; El Deeb, Sami; Wätzig, Hermann

    2015-03-25

    In this work, the behavior of several metal ions with different globular proteins was investigated by affinity capillary electrophoresis. Screening was conducted by applying a proper rinsing protocol developed by our group. The use of 0.1M EDTA in the rinsing solution successfully desorbs metal ions from the capillary wall. The mobility ratio was used to evaluate the precision of the method. Excellent precision for repeated runs was achieved for different protein metal ion interactions (RSD% of 0.05-1.0%). Run times were less than 6min for all of the investigated interactions. The method has been successfully applied for the interaction study of Li(+), Na(+), Mg(2+), Ca(2+), Ba(2+), Al(3+), Ga(3+), La(3+), Pd(2+), Ir(3+), Ru(3+), Rh(3+), Pt(2+), Pt(4+), Os(3+), Au(3+), Au(+), Ag(+), Cu(1+), Cu(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cr(3+), V(3+), MoO4(2-) and SeO3(2-) with bovine serum albumin, ovalbumin, ?-lactoglobulin and myoglobin. Different interaction values were obtained for most of the tested metal ions even for that in the same metal group. Results were discussed and compared in view of metal and semimetal group's interaction behavior with the tested proteins. The calculated normalized difference of mobility ratios for each protein-metal ion interaction and its sign (positive and negative) has been successfully used to detect the interaction and estimate further coordination of the bound metal ion, respectively. The comprehensive platform summarizes all the obtained interaction results, and is valuable for any future protein-metal ion investigation. PMID:25638307

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

    PubMed Central

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

    2009-01-01

    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. PMID:19411661

  19. Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis.

    PubMed

    Harder, A; Wildgruber, R; Nawrocki, A; Fey, S J; Larsen, P M; Görg, A

    1999-01-01

    Three different procedures for the solubilization of yeast (S. cerevisiae) cell proteins were compared on the basis of the obtained two-dimensional (2-D) polypeptide patterns. Major emphasis was laid on minimizing handling steps, protein modification or degradation, and quantitative loss of high molecular mass proteins. The procedures employed were sonication, followed by (i) protein solubilization with "standard" lysis buffer (9 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), 2% v/v carrier ampholytes, (ii) presolubilization of proteins with sodium dodecyl sulfate (SDS) buffer, consisting of 1% SDS and 100 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, pH 7.0, followed by dilution with "standard" lysis buffer, and (iii) boiling the sample with SDS during cell lysis, followed by dilution with thiourea/urea lysis buffer (2 M thiourea/ 7 M urea, 4% w/v CHAPS, 1% w/v DTT, 2% v/v carrier ampholytes). All procedures tested were rapid and simple. However, with the first procedure (i), considerable degradation of high Mr proteins occurred. In contrast, protein degradation was minimized by boiling the sample in SDS buffer immediately after sonication (method ii). Protein disaggregation and solubilization of high Mr proteins were further improved by pre-boiling with SDS and using thiourea/urea lysis buffer instead of "standard" lysis buffer (procedure iii). PMID:10344254

  20. A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

    SciTech Connect

    Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl; Dong, Ming; Biggin, Mark D.; Jin, Jian

    2009-10-02

    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 columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.

  1. Studies on proteinograms in dermatorphytes by disc electrophoresis. Part 2: Protein bands of keratinophilic fungi

    NASA Technical Reports Server (NTRS)

    Danev, P.; Balabanov, V.; Friedrich, E.

    1983-01-01

    Disc electrophoresis studies on keratinophili fungi demonstrated corresponding proteinograms in morphologically homogeneous strains of the same species, but different in different species of one and the same genus.

  2. Migration behaviour of discontinuous buffers in capillary electrophoresis during protein enrichment.

    PubMed

    Li, Ting; Booker, Christina J; Yeung, Ken K-C

    2012-10-21

    Capillary electrophoresis (CE) is not only an effective separation technique, but can also serve as a sample preparation tool for enrichment and purification at sub-microliter sample volumes. Our approach is based on the use of a discontinuous buffer system consisting of an acid and a base (acetate and ammonium). Proteins and/or peptides with isoelectric points between the pH values of these two buffers will become stacked at the neutralization reaction boundary (NRB). To understand the mechanism of the NRB formation and the electrophoretic migration of various ions during the enrichment, we performed experiments using myoglobin and mesityl oxide to reveal the ion migration patterns at the buffer junction, and utilized Simul 5 to computer simulate the process. The simulated results closely resembled the experimental data, and together, they effectively revealed the characteristics of the discontinuous buffers. Importantly, the discovery allowed the manipulation of NRB behaviours by controlling the discontinuous buffer composition. To illustrate this, the removal of urea as an unwanted background molecule from the enriched protein sample was achieved based on the acquired information. PMID:22919699

  3. Mapping of polar fox renal cortex proteins using two-dimensional gel electrophoresis and mass spectrometry--a preliminary study.

    PubMed

    Ciechanowicz, A K; Ozgo, M; Sta?ski, ? R; Herosimczyk, A; Piotrowska, A; Szymeczko, R; Laszczy?ska, M; Skrzypczak, W F

    2014-01-01

    The aim of the present study was to establish protein map of polar fox (Alopex lagopus) renal cortex. Kidney cortex proteins of isoelectric point ranging from 3 to 10 were analysed using two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Sixteen protein spots corresponding to thirteen different gene products were identified. These proteins were divided into following groups: lipid and fatty acid metabolism, amino acid metabolism, energetic pathways, regulatory proteins, transport proteins and structural proteins. This is the first attempt to create reproducible 2-D map, of renal cortex proteins characteristic for polar foxes, used as animal model for carnivores. It is worth emphasizing that the results of this study may broaden currently available protein databases. PMID:24988848

  4. Continuous Signal Enhancement for Sensitive Aptamer Affinity Probe Electrophoresis Assay Using Electrokinetic Concentration

    E-print Network

    Cheow, Lih Feng

    We describe an electrokinetic concentration-enhanced aptamer affinity probe electrophoresis assay to achieve highly sensitive and quantitative detection of protein targets in a microfluidic device. The key weaknesses of ...

  5. Analysis of the interaction between hyaluronan and hyaluronan-binding proteins by capillary affinity electrophoresis: significance of hyaluronan molecular size on binding reaction

    Microsoft Academic Search

    Mitsuhiro Kinoshita; Kazuaki Kakehi

    2005-01-01

    We developed a method for the analysis of the interaction between hyaluronan (HA) oligosaccharides and hyaluronan-binding proteins (HABPs) using capillary affinity electrophoresis (CAE). The method is based on high-resolution separation of fluorescent-labeled HA molecules in the presence of hyaluronan-binding proteins at different concentrations by capillary electrophoresis (CE) with laser-induced fluorescent detection. Hyaluronan-binding protein from bovine nasal cartilage interacts strongly with

  6. Precision in affinity capillary electrophoresis for drug-protein binding studies.

    PubMed

    El-Hady, Deia; Kühne, Sascha; El-Maali, Nagwa; Wätzig, Hermann

    2010-06-01

    In order to achieve excellent precision in the estimation of binding constants by affinity capillary electrophoresis (ACE), electroosmotic flow (EOF) stability is the key parameter, especially when using proteins in binding assays. Appropriate rinsing protocols are mandatory. In our study, the capillary was rinsed after each run with 0.1 mol/L sodium hydroxide for 2.0 min, with water for 2.0 min followed by running electrolyte (phosphate buffer at pH 7.4) for 3.0 min (pressure=3000 mbar each). Tryptophan-human serum albumin, warfarin-bovine serum albumin and quercetin-beta-lactoglobulin were used as ACE models. Further improvements in precision have been obtained by avoiding a complete standstill of liquid within the capillary and flushing the capillary with buffer for 25 min after each 30 consecutive runs. The precision of measurements is further improved by the use of mobility ratios to report mobility changes (RSD% less than 0.5% in a long-term measurement, n=300-600). Apart from the importance of a stable EOF, other ACE key parameters include protein concentration, drug plug length, applied voltage, and the choice of the regression method. In the present work, useful protocols and templates are provided in order to allow users a quick and efficient start with ACE methods. The comprehensive experimental part can serve as a checklist, which parameters need to be addressed for successfully applying ACE. Here, the suggested experimental design allows for the determination of binding constants within a couple of hours using standard instrumentation. This time could still be decreased by orders of magnitude using capillary arrays or miniaturized systems. PMID:20080373

  7. This work introduces a novel active contour-based scheme for unsupervised segmentation of protein spots in two-dimensional gel electrophoresis (2D-GE) images. The proposed segmentation scheme is the first to

    E-print Network

    Athens, University of

    of protein spots in two-dimensional gel electrophoresis (2D-GE) images. The proposed segmentation scheme: Segmentation; Active contours; 2D-gel electrophoresis images. * Corresponding author Tel.: +30-210-7275317 Fax-dimensional gel electrophoresis (2D-GE) [1]. In 2D-GE, an indicative portion of the total protein component

  8. A comparison of three serological assays, protein gel electrophoresis and the polymerase chain reaction for the detection of Chlomydia psittici infections in pet birds

    E-print Network

    Hofle, Michael David

    1999-01-01

    : complement fixation (CF), elementary body agglutination (EBA), immunofluorescent antibodies (IFA), polymerase chain reaction (PCR), protein gel electrophoresis. Comparison of the results obtained showed a strong statistical association between EBA to CF, PCR...

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

    SciTech Connect

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

    1985-05-01

    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.

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

    Microsoft Academic Search

    Philippe Delepelaire; Nam-Hai Chua

    1979-01-01

    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

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

    Microsoft Academic Search

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

    \\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

  12. Quantitative thermodynamic model for globular protein folding

    NASA Astrophysics Data System (ADS)

    Yakubovich, Alexander V.; Solov'yov, Andrey V.

    2014-06-01

    We present a statistical mechanics formalism for theoretical description of the process of protein folding ? unfolding transition in water environment. The formalism is based on the construction of the partition function of a protein obeying two-stage-like folding kinetics. Using the statistical mechanics model of solvation of hydrophobic hydrocarbons we obtain the partition function of infinitely diluted solution of proteins in water environment. The calculated dependencies of the protein heat capacities upon temperature are compared with the corresponding results of experimental measurements for staphylococcal nuclease and metmyoglobin.

  13. Proteins synthesized in African swine fever virus-infected cells analyzed by two-dimensional gel electrophoresis.

    PubMed

    Urzainqui, A; Tabarés, E; Carrasco, L

    1987-09-01

    At least 74 acidic and 37 basic proteins are synthesized in African swine fever virus (ASFV)-infected monkey cells not detected in uninfected cells analyzed by two-dimensional gel electrophoresis. Essentially all the proteins synthesized early during infection are also observed at late times. The use of inhibitors such as cycloheximide and phosphonoacetate has led to the identification of 34 immediate early and 13 delayed early polypeptides. Therefore 64 proteins were classified as late polypeptides. Several ASFV-induced proteins are phosphorylated as proteins a1, a4, a20, a41, a48, a49, a51, a52, a55, a58, a67, b2, b12, b28, and b32. PMID:3629977

  14. Comparative analysis of cellulose acetate hemoglobin electrophoresis and high performance liquid chromatography for quantitative determination of hemoglobin A2

    PubMed Central

    Khosa, Shafi Mohammad; Moinuddin, Moinuddin; Mehmood, Hassan Osman; Qamar, Khansa

    2015-01-01

    Background The present study is designed to evaluate the reliability and cost effectiveness of cellulose acetate Hb electrophoresis and high performance liquid chromatography (HPLC) in the determination of HbA2 levels. Methods The test population comprised 160 individuals divided into four groups: normal individuals, ?-thalassemia trait (BTT) patients, iron deficiency anemia (IDA) patients, and co-morbid patients (BTT with IDA). HbA2 levels determined using cellulose acetate Hb electrophoresis and HPLC were compared. Results HbA2 levels were found to be diagnostic for classical BTT using either method. In co-morbid cases, both techniques failed to diagnose all cases of BTT. The sensitivity, specificity, and Youden's index for detection of the co-morbid condition was 69% and 66% for HPLC and cellulose acetate Hb electrophoresis, respectively. Conclusion This study revealed that semi-automated cellulose acetate Hb electrophoresis is more suitable for use in ?-thalassemia prevention programs in low-income countries like Pakistan. This technique is easily available, simple and cost effective.

  15. Definition of Mycobacterium tuberculosis Culture Filtrate Proteins by Two-Dimensional Polyacrylamide Gel Electrophoresis, N-Terminal Amino Acid Sequencing, and Electrospray Mass Spectrometry

    Microsoft Academic Search

    MICHAEL G. SONNENBERG; JOHN T. BELISLE

    1997-01-01

    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

  16. Direct quantitative determination of amlodipine enantiomers in urine samples for pharmacokinetic study using on-line coupled isotachophoresis-capillary zone electrophoresis separation method with diode array detection.

    PubMed

    Miks, Peter; Maráková, Katarína; Marák, Jozef; Nemec, Igor; Valásková, Iva a; Havránek, Emil

    2008-11-01

    The present work illustrates possibilities of column-coupling capillary electrophoresis (CE-CE) combined with chiral selector (2-hydroxypropyl-beta-cyclodextrin, HP-beta-CD) and fiber-based diode array detection (DAD) for the direct quantitative enantioselective determination of trace drug (amlodipine, AML) in biological multicomponent ionic matrices (human urine). Capillary isotachophoresis (ITP) served as an ideal injection technique in CE-CE. Moreover, the ITP provided an effective on-line sample pretreatment prior to the capillary zone electrophoresis (CZE) separation. Enhanced separation selectivity due to the combination of different separation mechanisms (ITP vs. CZE-HP-beta-CD) enabled to obtain pure zones of the analytes, suitable for their detection and quantitation. The DAD, unlike single wavelength UV detection, enabled to characterize the purity (i.e. spectral homogeneity) of the analytes zones. A processing of the raw DAD spectra (the background correction and smoothing procedure) was essential when a trace analyte signal was evaluated. Obtained results indicated pure (i.e. spectrally homogeneous) zones of interest confirming effective ITP-CZE separation process. The proposed ITP-CZE-DAD method was characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, selectivity) and successfully applied to an enantioselective pharmacokinetic study of AML. PMID:18599368

  17. Colorful Electrophoresis

    NSDL National Science Digital Library

    2012-06-26

    In this activity, learners follow step-by-step instructions to build a gel electrophoresis chamber using inexpensive materials from local hardware and electronic stores. Then, learners follow instructions to simulate DNA electrophoresis using food colors from the kitchen pantry.

  18. Age-associated changes in synaptic lipid raft proteins revealed by two-dimensional fluorescence difference gel electrophoresis

    PubMed Central

    Jiang, Lei; Fang, Jianwen; Moore, David S.; Gogichaeva, Natalia V.; Galeva, Nadezhda A.; Michaelis, Mary L.; Zaidi, Asma

    2009-01-01

    Brain aging is associated with a progressive decline in cognitive function though the molecular mechanisms remain unknown. Functional changes in brain neurons could be due to age-related alterations in levels of specific proteins critical for information processing. Specialized membrane microdomains known as ‘lipid rafts’ contain protein complexes involved in many signal transduction processes. This study was undertaken to determine if two-dimensional fluorescence difference gel electrophoresis (2D DIGE) analysis of proteins in synaptic membrane lipid rafts revealed age-dependent alterations in levels of raft proteins. Five pairs of young and aged rat synaptic membrane rafts were subjected to DIGE separation, followed by image analysis and identification of significantly altered proteins. Of 1046 matched spots on DIGE gels, 94 showed statistically significant differences in levels between old and young rafts, and 87 of these were decreased in aged rafts. The 41 most significantly altered (p < 0.03) proteins included several synaptic proteins involved in energy metabolism, redox homeostasis, and cytoskeletal structure. This may indicate a disruption in bioenergetic balance and redox homeostasis in synaptic rafts with brain aging. Differential levels of representative identified proteins were confirmed by immunoblot analysis. Our findings provide novel pathways in investigations of mechanisms that may contribute to altered neuronal function in aging brain. PMID:19118924

  19. Characterization of proteins in latex of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis and microsequencing.

    PubMed

    Decker, G; Wanner, G; Zenk, M H; Lottspeich, F

    2000-10-01

    The opium poppy (Papaver somniferum) belongs to the group of latex-containing plants. Latex is the milky-like fluid within laticifer cells. In this study, poppy latex was analyzed with respect to ultrastructure, alkaloid, and protein content. The main goal of this project was the examination of the proteins by two-dimensional gel electrophoresis. In a proteomics approach, we investigated two main fractions of the latex, namely the cytosolic serum and the sedimented fraction containing the alkaloid-accumulating vesicles. Of the serum, representing the protein-rich part of the latex, 75 spots were analyzed by internal peptide microsequencing, followed by a database searching. For 69 proteins a function could be assigned due to homology to known proteins, whereas six spots could not be identified. Furthermore, codeinone reductase, a representative of the specific enzyme system in morphine biosynthesis, could be detected within the cytosolic serum fraction. In the vesicle-containing pellet, 23 protein spots were analyzed. An attempt was also made to separate the vesicle pellet by density centrifugation, followed by investigation of the alkaloid content, ultrastructure, and protein pattern. This study describes the first database of soluble proteins present in the latex of P. somniferum PMID:11079569

  20. Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis

    SciTech Connect

    Carrasco, L.; Bravo, R.

    1986-05-01

    The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various time postinfection were also analyzed. At least 13 proteins labeled with (/sup 3/H)glucosamine were detected in vaccinia-infected HeLa cells.

  1. Capillary electrophoresis for clinical problem solving: analysis of urinary diagnostic metabolites and serum proteins

    Microsoft Academic Search

    Egil Jellum; Herman Dollekamp; Cynthia Blessum

    1996-01-01

    Many clinical laboratories employ gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) to detect abnormal compounds occurinng in urine and serum due to disease. The methods, particularly GC-MS, often require laborious sample pre-treatment, and separation times may exceed an hour. We describe the use of capillary electrophoresis (CE) equipped a with a diode-array detector in an attempt to improve

  2. New ways in qualitative and quantitative protein analysis: nano chromatography coupled to element mass spectrometry.

    PubMed

    Schaumlöffel, Dirk

    2007-01-01

    The potential of inductively coupled plasma-mass spectrometry (ICP-MS), which allows element-specific detection of heteroelements (e.g. Se and S) incorporated in protein structures, is highlighted for sensitive qualitative and quantitative protein analysis. ICP-MS coupled to separation techniques such as size exclusion chromatography and gel electrophoresis (via laser ablation) can be employed at different steps in the proteomic workflow. Special emphasis is made on the couplings of capillary and nanoHPLC to ICP-MS that required the development of dedicated interfaces. Element-specific peptide mapping by nanoHPLC-ICP-MS has turned out to be a key technique in combination with peptide sequencing via nanoHPLC-electrospray MS. This could impressively be demonstrated for the identification of selenium-containing proteins in selenium-rich yeast. Furthermore the potential of sulfur isotope dilution analysis in nanoHPLC-ICP-MS is presented as generic tool for highly accurate, absolute protein quantification. PMID:18039489

  3. Proteomic analysis of surface proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry

    PubMed Central

    2013-01-01

    Background Trichinella spiralis is a zoonotic tissue-dwelling parasitic nematode that infects humans and other mammals. Its surface proteins are recognized as antigenic in many infected hosts, being directly exposed to the host’s immune system and are the main target antigens that induce the immune responses. The larval surface proteins may also interact with intestinal epithelial cells and may play an important role in the invasion and development process of T. spiralis. The purpose of this study was to analyze and characterize the surface proteins of T. spiralis muscle larvae by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Methods The surface proteins of T. spiralis muscle larvae were stripped from the cuticle of live larvae by the cetyltrimethylammonium bromide (CTAB) and sodium deoxycholate. The surface protein stripping was examined by an immunofluorescent test (IFT). The surface proteins were analyzed by SDS-PAGE and Western blotting, and then identified by 2-DE and MALDI-TOF/TOF mass spectrometry analysis. Results The IFT results showed that the surface proteins-stripped larvae were not recognized by sera of mice immunized with surface antigens. Western blotting showed 7 of 12 protein bands of the surface proteins were recognized by mouse infection sera at 18 dpi and at 42 dpi. The 2-DE results showed that a total of approximately 33 proteins spots were detected with molecular weights varying from 10 to 66 kDa and isoelectric point (pI) from 4 to 7. Twenty-seven of 33 protein spots were identified and characterized to correlate with 15 different proteins. Out of the 14 proteins identified as T. spiralis proteins, 5 proteins (partial P49 antigen, deoxyribonuclease II family protein, two serine proteases, and serine proteinase) had catalytic and hydrolase activity. All of these 5 proteins were also associated with metabolic processes and 2 of the five proteins were associated with cellular processes. Conclusions In this study, T. spiralis muscle larval surface proteins have been identified, which will provide useful information to elucidate the host-parasite interaction, identify the invasion-related proteins, early diagnostic antigens and the targets for a vaccine. PMID:24330777

  4. Proteomics analysis in mature seed of four peanut cultivars using two-dimensional gel electrophoresis reveals distinct differential expression of storage, anti-nutritive, and allergenic proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein profiles of total seed proteins isolated from mature seeds of four peanut cultivars, New Mexico Valencia C (NM Valencia C), Tamspan 90, Georgia Green, and NC-7, were studied using two-dimensional gel electrophoresis coupled with nano electrospray ionization liquid chromatography tandem mass ...

  5. Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

    Cancer.gov

    In cooperation with the RAS Initiative, the NCI's Clinical Proteomic Tumor Analysis Consortium (CPTAC) has launched a project to develop quantitative assays for proteins and phosphopeptides involved in RAS signaling. Within the next 1-2 years these assays should allow the amounts and phosphorylation states of tens of RAS and RAS-related proteins to be determined in tumor samples, cell lines, or cancer models in a single run.

  6. Protein identification from two-dimensional gel electrophoresis analysis of Klebsiella pneumoniae by combined use of mass spectrometry data and raw genome sequences

    Microsoft Academic Search

    Wei Wang; Jibin Sun; Manfred Nimtz; Wolf-Dieter Deckwer; An-Ping Zeng

    2003-01-01

    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

  7. A versatile electrophoresis system for the analysis of high and low molecular weight proteins Christophe Tastet1, Pierre Lescuyer1, Hlne Diemer2, Sylvie Luche1, Alain van Dorsselaer2

    E-print Network

    Paris-Sud XI, Université de

    A versatile electrophoresis system for the analysis of high and low molecular weight proteins sodium dodecyl sulfate- polyacrylamide gel electrophoresis of proteins in the relative molecular weight-20 kDa, its performances decreases for proteins of lower molecular weight. To be analyzed

  8. A Quantitative Assessment of Heterogeneity for Surface-Immobilized Proteins

    E-print Network

    Leckband, Deborah E.

    A Quantitative Assessment of Heterogeneity for Surface-Immobilized Proteins Ravi A. Vijayendran strategies. We used the Sips isotherm to assesses and compare the heterogeneity in the antibody binding directly on a solid substrate or within a porous matrix and used to detect the presence of a target

  9. Identification of SUMO target proteins by quantitative proteomics.

    PubMed

    Andersen, Jens S; Matic, Ivan; Vertegaal, Alfred C O

    2009-01-01

    The identification of target proteins for small ubiquitin-like modifiers (SUMOs) is a critical step towards a detailed understanding of the cellular functions of SUMOs. Substrate protein identification for SUMOs is hampered by the low abundance of SUMO targets, the finding that only a small fraction of these target proteins is sumoylated, and the high activity of deconjugating enzymes. Quantitative proteomics is a powerful tool to overcome these challenges, because it allows discrimination between contaminating proteins in SUMO-enriched preparations and true target proteins. In this chapter, the methodological details of the application of stable isotope labeling of amino acids in cell culture (SILAC) for the identification of target proteins for SUMOs are described. In addition to steady state sumoylation, the sumoylated proteome undergoes dynamic rearrangements in response to a diverse array of stimuli. SILAC also allows the study of sumoylation dynamics in response to these stimuli. PMID:19107408

  10. Quantitating metabolites in protein precipitated serum using NMR spectroscopy.

    PubMed

    Nagana Gowda, G A; Raftery, Daniel

    2014-06-01

    Quantitative NMR-based metabolite profiling is challenged by the deleterious effects of abundant proteins in the intact blood plasma/serum, which underscores the need for alternative approaches. Protein removal by ultrafiltration using low molecular weight cutoff filters thus represents an important step. However, protein precipitation, an alternative and simple approach for protein removal, lacks detailed quantitative assessment for use in NMR based metabolomics. In this study, we have comprehensively evaluated the performance of protein precipitation using methanol, acetonitrile, perchloric acid, and trichloroacetic acid and ultrafiltration approaches using 1D and 2D NMR, based on the identification and absolute quantitation of 44 human blood metabolites, including a few identified for the first time in the NMR spectra of human serum. We also investigated the use of a "smart isotope tag," (15)N-cholamine for further resolution enhancement, which resulted in the detection of a number of additional metabolites. (1)H NMR of both protein precipitated and ultrafiltered serum detected all 44 metabolites with comparable reproducibility (average CV, 3.7% for precipitation; 3.6% for filtration). However, nearly half of the quantified metabolites in ultrafiltered serum exhibited 10-74% lower concentrations; specifically, tryptophan, benzoate, and 2-oxoisocaproate showed much lower concentrations compared to protein precipitated serum. These results indicate that protein precipitation using methanol offers a reliable approach for routine NMR-based metabolomics of human blood serum/plasma and should be considered as an alternative to ultrafiltration. Importantly, protein precipitation, which is commonly used by mass spectrometry (MS), promises avenues for direct comparison and correlation of metabolite data obtained from the two analytical platforms to exploit their combined strength in the metabolomics of blood. PMID:24796490

  11. Quantitative description of analyte migration behavior based on dynamic complexation in capillary electrophoresis with one or more additives.

    PubMed

    Peng, X; Bowser, M T; Britz-McKibbin, P; Bebault, G M; Morris, J R; Chen, D D

    1997-05-01

    A comprehensive theory is proposed to describe the migration behavior of analytes in capillary electrophoresis (CE) when one or more additives are present in the buffer solution. This theory amalgamates and extends the previous work done by others. The capacity factor (k') in this theory is defined as the product of the equilibrium constant and the additive concentration, thus, k' changes linearly with additive concentration. The net electrophoretic mobility of an analyte is a function of k', therefore, it can be changed by varying the additive concentration. Three parameters are needed to predict the mobility of an analyte in a one-additive CE system: the mobility of the free analyte, the mobility of the complex, and the equilibrium constant for the analyte-additive interaction (which determines the fraction of the free analyte at different additive concentrations). When additives are used, the change in viscosity obscures this relationship, therefore, a viscosity correction factor is required to convert all mobilities to an ideal state where the viscosity remains constant. The migration behavior of an analyte in a solution with multiple additives can be predicted and controlled, once the equilibrium constants of the interactions between the analyte and each of the additives are obtained separately. beta-Cyclodextrin and hydroxypropyl-beta-cyclodextrin are used as additives and the migration behavior of phenol, p-nitrophenol, and benzoic acid are studied as a model system to verify this theory. When the necessary viscosity correction factor is included, the net electrophoretic mobilities of the analytes obtained from experimental results agree with the values predicted by the theory based on dynamic complexation. Although only experiments with one and two additives were carried out to verify the theory, the equations apply to situations when more than two additives are used. The relationship between the theories of electrophoresis and chromatography is clarified. PMID:9194595

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

    Microsoft Academic Search

    B. C. Johnson; A. M. Swanson

    1952-01-01

    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

  13. Attomole sensitivity for unlabeled proteins and polypeptides with on-chip capillary electrophoresis and universal detection by interferometric backscatter.

    PubMed

    Wang, Zhanling; Swinney, Kelly; Bornhop, Darryl J

    2003-03-01

    A universal detector based on backscatter interferometry has been developed to perform nanoliter volume refractive index measurements for on-chip sodium dodecyl sulfate (SDS) gel based (polyethylene oxide gel) separations and quantification label-free proteins. The on-chip interferometric backscatter detector (OCIBD) system consists of a simple, folded optical train based on the interaction of a laser beam with an etched channel in the shape of half cylinder in a fused-silica plate. The backscattered light from the channel takes on the form of a high-contrast interference pattern that contains information related to the bulk properties of the fluid located within the probe or detection volume of 2.32 x 10(-9) L. Depending on capillary electrophoresis (CE) injection method, the positional changes of the interference pattern extrema (fringes) allow for the quantification of unlabeled proteins at levels ranging from 11 to 310 amol (2.7 x 10(-8)mol/L) with a linear dynamic range of 2.5 decades (egg albumin). Using OCIBD microchannel-based SDS capillary gel electrophoresis (SDS/CGE), separation and detection of five label-free proteins was achieved in less than 100 seconds with detection limits ranging from 0.95 pg (1.1 x 10(-16)mol or 2.5 x 10(-7)mol/L) of calmodulin to 7.0 pg (1.0 x 10(-16)mol or 2.4 x 10(-7)mol/L) for bovine serum albumin (BSA) without signal filtering or active thermal control. This development shows that a universal detector based on backscatter interferometry can be used effectively for on-chip label-free solute analysis. PMID:12627449

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

    Microsoft Academic Search

    Chenping Xu; Yan Xu; Bingru Huang

    2008-01-01

    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,

  15. Quantitative Protein Localization Signatures Reveal an Association between Spatial and Functional Divergences of Proteins

    PubMed Central

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-01-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein functions and how these functions were acquired in cells from different organisms or species. A public web interface of PLAST is available at http://plast.bii.a-star.edu.sg. PMID:24603469

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

    Microsoft Academic Search

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

    2011-01-01

    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

  17. Quantitation of talinolol and other beta-blockers by capillary electrophoresis for in vitro drug absorption studies.

    PubMed

    Awadallah, Bilal; Schmidt, Peter C; Holzgrabe, Ulrike; Wahl, Martin A

    2003-08-01

    A capillary zone electrophoresis method is described for the enantioseparation of talinolol using heptakis(2,3-diacetyl-6-sulfo)-beta-cyclodextrin (HDAS-beta-CD) as a chiral selector. After liquid-liquid extraction of talinolol from physiological solution, electrokinetic injection was employed to improve the sensitivity. The use of a coated capillary was necessary to achieve stable and reproducible enantioseparations. A baseline separation of the talinolol enantiomers was achieved in less than 10 min using 100 mM phosphate solution as background electrolyte and pH 3.5, at the presence of 3.0 mM HDAS-beta-CD and at 20 degrees C. In addition, this analytical condition proved to be useful for the enantioseparation of a number of other beta-blocking agents such as alprenolol, atenolol, bisoprolol, celiprolol, metipranolol, oxprenolol, and sotalol. For determining talinolol, the method could be validated in terms of precision, accuracy and linearity, and was found to be suitable in determination of talinolol enantiomers in highly diluted samples obtained from in vitro experiments. PMID:12900875

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

    SciTech Connect

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

    1987-05-01

    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.

  19. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    NASA Technical Reports Server (NTRS)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

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

    PubMed Central

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

    2013-01-01

    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. PMID:24058531

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat flour is one of the world's major food ingredients, but it is difficult to distinguish and identify the many proteins in a flour sample. The abundant glutamine and proline rich gluten proteins are responsible for many of the unique end-use qualities of wheat flour but it is challenging to dis...

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

    PubMed

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

    2013-11-01

    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

  3. Fusion-Related Host Proteins Are Actively Regulated by NA during Influenza Infection as Revealed by Quantitative Proteomics Analysis

    PubMed Central

    Sui, Zhiwei; Wen, Bo; Gao, Zhimin; Chen, Quanjiao

    2014-01-01

    Three recombinant influenza A viruses with different neuraminidases (NAs) in the background of A/PR/8/34 (PR8), named rPR8-H5N1NA, rPR8-H9N2NA, and rPR8-H1N1NA, derived from H5N1, H9N2, H1N1 (swine) viruses, respectively, were constructed. We performed a quantitative proteomics analysis to investigate differential protein expression in Madin-Darby canine kidney (MDCK) cells infected with recombinant and wild-type influenza viruses to determine whether NA replacement would alter host cell gene expression. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS) and two-dimensional gel electrophoresis (2-DE), we identified 12 up-regulated and 49 down-regulated protein spots, including cytoskeletal proteins, molecular biosynthesis proteins, ubiquitin-proteasome pathway proteins, and heat shock proteins. The most significant changes in infected cells were observed for molecular biosynthesis proteins. We found more differentially expressed protein spots in cells infected with rPR8-H5N1NA or rPR8-H9N2NA viruses than cells infected with wild-type virus. Many of those proteins are postulated to be involved in cell-cell fusion, but the full mechanism remains to be explored. Meanwhile, our data demonstrate that the wild-type virus has evolutionary advantages over recombinant viruses. PMID:25153908

  4. Evaluating two-dimensional electrophoresis profiles of the protein phaseolin as markers of genetic differentiation and seed protein quality in common bean (Phaseolus vulgaris L.).

    PubMed

    López-Pedrouso, María; Bernal, Javier; Franco, Daniel; Zapata, Carlos

    2014-07-23

    High-resolution two-dimensional electrophoresis (2-DE) profiles of the protein phaseolin, the major seed storage protein of common bean, display great number of spots with differentially glycosylated and phosphorylated ?- and ?-type polypeptides. This work aims to test whether these complex profiles can be useful markers of genetic differentiation and seed protein quality in bean populations. The 2-DE phaseolin profile and the amino acid composition were examined in bean seeds from 18 domesticated and wild accessions belonging to the Mesoamerican and Andean gene pools. We found that proteomic distances based on 2-DE profiles were successful in identifying the accessions belonging to each gene pool and outliers distantly related. In addition, accessions identified as outliers from proteomic distances showed the highest levels of methionine content, an essential amino acid deficient in bean seeds. These findings suggest that 2-DE phaseolin profiles provide valuable information with potential of being used in common bean genetic improvement. PMID:24983510

  5. Adaption of a fragment analysis technique to an automated high-throughput multicapillary electrophoresis device for the precise qualitative and quantitative characterization of microbial communities.

    PubMed

    Trotha, René; Reichl, Udo; Thies, Frank L; Sperling, Danuta; König, Wolfgang; König, Brigitte

    2002-04-01

    The analysis of microbial communities is of increasing importance in life sciences and bioengineering. Traditional techniques of investigations like culture or cloning methods suffer from many disadvantages. They are unable to give a complete qualitative and quantitative view of the total amount of microorganisms themselves, their interactions among each other and with their environment. Obviously, the determination of static or dynamic balances among microorganisms is of fast growing interest. The generation of species specific and fluorescently labeled 16S ribosomal DNA (rDNA) fragments by the terminal restriction fragment length polymorphism (T-RFLP) technique is a suitable tool to overcome the problems other methods have. For the separation of these fragments polyacrylamide gel sequencers are preferred as compared to capillary sequencers using linear polymers until now because of their higher electrophoretic resolution and therefore sizing accuracy. But modern capillary sequencers, especially multicapillary sequencers, offer an advanced grade of automation and an increased throughput necessary for the investigation of complex communities in long-time studies. Therefore, we adapted a T-RFLP technique to an automated high-throughput multicapillary electrophoresis device (ABI 3100 Genetic Analysis) with regard to a precise qualitative and quantitative characterization of microbial communities. PMID:11981854

  6. Towards quantitative prediction of proteasomal digestion patterns of proteins

    E-print Network

    Goldobin, Denis S

    2014-01-01

    We discuss the problem of proteasomal degradation of proteins. Though proteasomes are important for all aspects of the cellular metabolism, some details of the physical mechanism of the process remain unknown. We introduce a stochastic model of the proteasomal degradation of proteins, which accounts for the protein translocation and the topology of the positioning of cleavage centers of a proteasome from first principles. For this model we develop the mathematical description based on a master-equation and techniques for reconstruction of the cleavage specificity inherent to proteins and the proteasomal translocation rates, which are a property of the proteasome specie, from mass spectroscopy data on digestion patterns. With these properties determined, one can quantitatively predict digestion patterns for new experimental set-ups. Additionally we design an experimental set-up for a synthetic polypeptide with a periodic sequence of amino acids, which enables especially reliable determination of translocation ...

  7. Towards quantitative prediction of proteasomal digestion patterns of proteins

    E-print Network

    Denis S. Goldobin; Alexey Zaikin

    2008-06-16

    We discuss the problem of proteasomal degradation of proteins. Though proteasomes are important for all aspects of the cellular metabolism, some details of the physical mechanism of the process remain unknown. We introduce a stochastic model of the proteasomal degradation of proteins, which accounts for the protein translocation and the topology of the positioning of cleavage centers of a proteasome from first principles. For this model we develop the mathematical description based on a master-equation and techniques for reconstruction of the cleavage specificity inherent to proteins and the proteasomal translocation rates, which are a property of the proteasome specie, from mass spectroscopy data on digestion patterns. With these properties determined, one can quantitatively predict digestion patterns for new experimental set-ups. Additionally we design an experimental set-up for a synthetic polypeptide with a periodic sequence of amino acids, which enables especially reliable determination of translocation rates.

  8. Study of Early Leaf Senescence in Arabidopsis thaliana by Quantitative Proteomics Using Reciprocal 14N\\/15N Labeling and Difference Gel Electrophoresis

    Microsoft Academic Search

    Romano Hebeler; Silke Oeljeklaus; Kai A. Reidegeld; Martin Eisenacher; Christian Stephan; Barbara Sitek; Kai Stuhler; Helmut E. Meyer; Marcel J. G. Sturre; Paul P. Dijkwel; Bettina Warscheid

    2007-01-01

    Leaf senescence represents the final stage of leaf develop- ment and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senes- cence, we designed an elaborate double and reverse label- ing strategy simultaneously employing fluorescent two-di- mensional DIGE as well as metabolic 15N labeling followed

  9. Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics*

    PubMed Central

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

    2013-01-01

    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. PMID:23242552

  10. A visual detection of protein content based on titration of moving reaction boundary electrophoresis.

    PubMed

    Wang, Hou-Yu; Guo, Cheng-Ye; Guo, Chen-Gang; Fan, Liu-Yin; Zhang, Lei; Cao, Cheng-Xi

    2013-04-24

    A visual electrophoretic titration method was firstly developed from the concept of moving reaction boundary (MRB) for protein content analysis. In the developed method, when the voltage was applied, the hydroxide ions in the cathodic vessel moved towards the anode, and neutralized the carboxyl groups of protein immobilized via highly cross-linked polyacrylamide gel (PAG), generating a MRB between the alkali and the immobilized protein. The boundary moving velocity (V(MRB)) was as a function of protein content, and an acid-base indicator was used to denote the boundary displacement. As a proof of concept, standard model proteins and biological samples were chosen for the experiments to study the feasibility of the developed method. The experiments revealed that good linear calibration functions between V(MRB) and protein content (correlation coefficients R>0.98). The experiments further demonstrated the following merits of developed method: (1) weak influence of non-protein nitrogen additives (e.g., melamine) adulterated in protein samples, (2) good agreement with the classic Kjeldahl method (R=0.9945), (3) fast measuring speed in total protein analysis of large samples from the same source, and (4) low limit of detection (0.02-0.15 mg mL(-1) for protein content), good precision (R.S.D. of intra-day less than 1.7% and inter-day less than 2.7%), and high recoveries (105-107%). PMID:23567122

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

    PubMed

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

    2010-01-01

    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. PMID:20440404

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

    PubMed Central

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

    2010-01-01

    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. PMID:20440404

  13. Structural characterization of polypeptides and proteins by combination of capillary electrophoresis and (252)Cf plasma desorption mass spectrometry.

    PubMed

    Weinmann, W; Baumeister, K; Kaufmann, I; Przybylski, M

    1993-01-01

    An efficient and sensitive method for the isolation and transfer of peptides and proteins from capillary zone electrophoresis separation for subsequent analysis by 252Cf plasma desorption mass spectrometry was developed. Sample isolation on to nitrocellulose-coated targets for mass spectrometric analysis is performed by using a stainless-steel microtube pre-filled with aqueous buffer solution, to which the capillary end is connected, and the peptide is collected by applying a suitable transfer voltage according to the separation voltage. Low-and sub-picomolar sample amounts were isolated with high transfer efficiency and reproducibility, without the necessity for independent determination of electroosmotic flow-rates. Plasma desorption mass spectra of several peptides and proteins showed predominantly intact molecular ions; however, for several peptides partial oxidative modification was found which can be accounted for by the electrophoretic separation and/or transfer conditions. First applications to peptides and proteins show the feasibility of this off-line combination for primary structure characterization, such as by in situ chemical modification and enzymatic proteolysis reactions on the sample target prior to mass spectrometric analysis. PMID:8429073

  14. An Integrated Quantitative Proteomics and Systems Biology Approach to Explore Synaptic Protein Profile Changes During Morphine Exposure

    PubMed Central

    Stockton, Steven D; Devi, Lakshmi A

    2014-01-01

    Morphine is a classic analgesic for the treatment of chronic pain. However, its repeated use is known to produce tolerance, physical dependence, and addiction; these properties limit its long-term therapeutic use and this has led to a quest for therapeutics without these unwanted side effects. Understanding the molecular changes in response to long-term use of morphine is likely to aid in the development of novel therapeutics for the treatment of pain. Studies examining the effects of chronic morphine administration have reported alterations in gene expression, synapse morphology, and synaptic transmission implying changes in synaptic protein profile. To fully understand the changes in protein profiles, proteomic techniques have been used. Studies using two-dimensional gel electrophoresis of various brain regions combined with mass spectrometry have found alterations in the levels of a number of proteins. However, neither the changes in brain regions relevant to morphine effects nor changes in the abundance of synaptic proteins have been clearly delineated. Recent studies employing subcellular fractionation to isolate the striatal synapse, combined with quantitative proteomics and graph theory-inspired network analyses, have begun to quantify morphine-regulated changes in synaptic proteins and facilitate the generation of networks that could serve as targets for the development of novel therapeutics for the treatment of chronic pain. Thus, an integrated quantitative proteomics and systems biology approach can be useful to identify novel targets for the treatment of pain and other disorders of the brain. PMID:24045585

  15. Electrophoresis. Author manuscript A versatile electrophoresis system for the analysis of high-and

    E-print Network

    Paris-Sud XI, Université de

    sulfatepolyacrylamide gel electrophoresis of proteins in the relative molecular weight Mw range of 300,000-3000 Da processes. MESH Keywords Buffers ; Electrophoresis, Gel, Two-Dimensional ; methods ; Electrophoresis weight. To be analyzed with this electrophoresis system, high concentration gels are needed (example

  16. Electrophoresis and isoelectric focusing of whole cell and membrane proteins from the extremely halophilic archaebacteria

    NASA Technical Reports Server (NTRS)

    Stan-Lotter, Helga; Lang, Frank J., Jr.; Hochstein, Lawrence I.

    1989-01-01

    The subunits from two purified halobacterial membrane enzymes (ATPase and nitrate reductase) behaved differently with respect to isoelectric focusing, silver staining and interaction with ampholytes. Differential behavior was also observed in whole cell proteins from Halobacterium saccharovorum regarding resolution in two-dimensional gels and silver staining. It is proposed that these differences reflect the existence of two classes of halobacterial proteins.

  17. Acid and Base-Induced Proteins during Aerobic and Anaerobic Growth of Escherichia coli Revealed by Two-Dimensional Gel Electrophoresis

    Microsoft Academic Search

    DARCY BLANKENHORN; JUDITH PHILLIPS; JOAN L. SLONCZEWSKI

    1999-01-01

    Proteins induced by acid or base, during long-term aerobic or anaerobic growth in complex medium, were identified in Escherichia coli. Two-dimensional gel electrophoresis revealed pH-dependent induction of 18 pro- teins, nine of which were identified by N-terminal sequencing. At pH 9, tryptophan deaminase (TnaA) was induced to a high level, becoming one of the most abundant proteins observed. TnaA may

  18. Multi-dimension microchip-capillary electrophoresis device for determination of functional proteins in infant milk formula.

    PubMed

    Wu, Ruige; Wang, Zhiping; Zhao, Wenfeng; Yeung, William Shu-Biu; Fung, Ying Sing

    2013-08-23

    To improve resolution of important minor proteins and eliminate time-consuming precipitation of major protein with associated analyte co-precipitation risk, a multi-dimension strategy is adopted in the 2D microchip-CE device to isolate major proteins on-chip, enrich minor proteins in capillary before their separation in CE for UV quantitation. A standard fluorescent protein mixture containing FITC-BSA, myoglobin and cytochrome as specific pI markers has prepared to demonstrate capability of the device to fractionate minor proteins by IEF. The results using a standard protein mixture with profile resembling infant milk formula show a complete isolation of high abundance proteins by a 2-min 1D IEF run. The subsequent t-ITP/CZE run by on-chip high voltage switching delivers a high stacking ratio, realizing 60 folds enrichment of isolated protein fractions. All five important functional proteins (LF, IgG, ?-LA, ?-LgA and ?-LgB) known to fortify infant milk formula are isolated and determined using two consecutive t-ITP-CZE runs within a 18-min total assay time, a significant saving compared to several hours conventional pretreatment. For a 100g infant milk formula sample, working ranges of 20-8000mg, repeatability 3.8-5.3% and detection limits 2.3-10mg have been achieved to meet government regulations. Method reliability is established by 100% recoveries and agreeable results within expected ranges and labeled values. The capability of the device for field operation, rapid assay with quick results, label-free universal detection, simple operation by aqueous dissolution before injection, and the demanding matching in 2D separation based on isolated fractions at specified pI ranges, closely matched migration time and baseline-resolved peak shape makes the device a general tool to detect unknown proteins and determine known minor proteins in protein-rich samples with interfering constituents. PMID:23870546

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

    NSDL National Science Digital Library

    Sean Sanders (Science/AAAS; )

    2012-11-14

    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.

  20. Mass spectrometric protein structure characterization reveals cause of migration differences of haptoglobin alpha chains in two-dimensional gel electrophoresis.

    PubMed

    Mikkat, Stefan; Koy, Cornelia; Ulbrich, Markus; Ringel, Bruno; Glocker, Michael O

    2004-12-01

    Haptoglobin belongs to the major constituents of plasma and acts as hemoglobin-binding and acute-phase protein. Due to the occurrence of three major allelic variants and further structural modifications, the alpha chains of haptoglobin form varying spot patterns in two-dimensional gel electrophoresis (2-DE) gels, which is generally observed in differential proteome analyses using plasma or related body fluids of humans. In the present study plasma samples from 10 donors of initially unknown haptoglobin phenotype were separated by 2-DE and tryptic digests of excised haptoglobin alpha chain spots were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and MALDI-quadrupole ion trap TOF-MS. Haptoglobin alpha1S, alpha1F, as well as alpha2 chains were found to occur each with at least three structurally differing protein species: (i) the unmodified form, which corresponds to the sequence database entries; (ii) derivatives, in which asparagine at position five is deamidated to aspartic acid; and (iii) derivatives with an additional C-terminal arginine residue. These structural variants account for the most commonly observed spot patterns of haptoglobin alpha chains in Coomassie-stained gels. Additionally, a minor derivative of the haptoglobin alpha2 chain carrying both modifications, deamidation at position five and the C-terminal arginine residue, was identified. Theoretical pI values of the characterized structural variants are, consistent with their observed migration in the 2-DE gels. PMID:15378693

  1. Gel Electrophoresis

    NSDL National Science Digital Library

    2007-04-19

    This interactive activity from the Dolan DNA Learning Center illustrates the process of gel electrophoresis, in which DNA fragments are separated by size as they migrate at different rates through a gel matrix.

  2. Application of two-dimensional gel electrophoresis in the study of cytoskeletal protein regulation during growth activation and differentiation.

    PubMed

    Ben-Ze'ev, A

    1990-03-01

    Two-dimensional gel electrophoresis was used to study the regulation of cytoskeletal protein synthesis during growth activation and development of the differentiated phenotype. We demonstrated a correlation between the state of organization and the expression of the respective cytoskeletal protein by showing that depolymerization of microtubules leads to a rapid decrease in new tubulin synthesis. We found that the synthesis of vimentin in both fibroblasts and epithelial cells correlates with extensive cell spreading on the substrate, while cytokeratin synthesis is maximal when cell to cell contacts are abundant. The analysis of cytoskeletal elements, involved directly in the formation of cell contacts, revealed that the level of vinculin synthesis is dependent on the extent of adherent type of cell contacts formed. Moreover, we found that the transient disappearance of vinculin from adhesion plaques of quiescent fibroblasts in response to serum factors was followed by an induction of vinculin mRNA and protein synthesis. The morphological changes associated with establishment of the differentiated phenotype were also found to include changes in the expression of the cytoskeletal-extracellular matrix complex. This was demonstrated in several differentiating systems: in 3T3 preadipocytes which change their shape from a fibroblastic to a spherical shape when stimulated to differentiate with adipogenic medium, we observed a decrease in mRNA levels and in the synthesis of fibronectin, beta-integrin, and the microfilament proteins, vinculin, alpha-actinin, tropomyosin and actin. The culturing of these cells on a certain extracellular matrix prevented the morphological changes occurring in the presence of adipogenic medium and blocked the shifts in cytoskeletal- and differentiation-related gene expression. Similar changes in the organization and expression of cytoskeletal proteins were identified during maturation of primary ovarian granulosa cell cultures, stimulated with gonadotropic hormones to form highly steroidogenic cells. The cell rounding and aggregation occurring during this process were associated with a decreased synthesis of vinculin, alpha-actinin, actin and the nonmuscle tropomyosins. The physiological relevance of these changes was suggested by the observation that the level of tropomyosin mRNA was lower in follicles of animals at late stages of granulosa cell maturation when compared to earlier stages. The expression of tissue-specific and cytoskeletal proteins was also determined in primary cultures of liver hepatocytes, maintained under conditions either favorable for growth or for expression of liver-specific functions. When DNA synthesis was elevated, cytoskeletal protein synthesis was high and that of liver-specific proteins was low.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2188832

  3. Gel Electrophoresis

    NSDL National Science Digital Library

    In the early days of DNA manipulation, DNA fragments were laboriously separated by gravity. In the 1970s, the powerful tool of DNA gel electrophoresis was developed. This process uses electricity to separate DNA fragments by size as they migrate through a gel matrix. This animation from Cold Spring Harbor Laboratory's Dolan DNA Learning Center presents Gel Electrophoresis through a series of illustrations of the processes involved.

  4. Binary Oscillatory Crossflow Electrophoresis

    NASA Technical Reports Server (NTRS)

    Molloy, Richard F.; Gallagher, Christopher T.; Leighton, David T., Jr.

    1997-01-01

    Electrophoresis has long been recognized as an effective analytic technique for the separation of proteins and other charged species, however attempts at scaling up to accommodate commercial volumes have met with limited success. In this report we describe a novel electrophoretic separation technique - Binary Oscillatory Crossflow Electrophoresis (BOCE). Numerical simulations indicate that the technique has the potential for preparative scale throughputs with high resolution, while simultaneously avoiding many problems common to conventional electrophoresis. The technique utilizes the interaction of an oscillatory electric field and a transverse oscillatory shear flow to create an active binary filter for the separation of charged protein species. An oscillatory electric field is applied across the narrow gap of a rectangular channel inducing a periodic motion of charged protein species. The amplitude of this motion depends on the dimensionless electrophoretic mobility, alpha = E(sub o)mu/(omega)d, where E(sub o) is the amplitude of the electric field oscillations, mu is the dimensional mobility, omega is the angular frequency of oscillation and d is the channel gap width. An oscillatory shear flow is induced along the length of the channel resulting in the separation of species with different mobilities. We present a model that predicts the oscillatory behavior of charged species and allows estimation of both the magnitude of the induced convective velocity and the effective diffusivity as a function of a in infinitely long channels. Numerical results indicate that in addition to the mobility dependence, the steady state behavior of solute species may be strongly affected by oscillating fluid into and out of the active electric field region at the ends of the cell. The effect is most pronounced using time dependent shear flows of the same frequency (cos((omega)t)) flow mode) as the electric field oscillations. Under such conditions, experiments indicate that solute is drawn into the cell from reservoirs at both ends of the cell leading to a large mass build up. As a consequence, any initially induced mass flux will vanish after short times. This effect was not captured by the infinite channel model and hence numerical and experimental results deviated significantly. The revised model including finite cell lengths and reservoir volumes allowed quantitative predictions of the time history of the concentration profile throughout the system. This latter model accurately describes the fluxes observed for both oscillatory flow modes in experiments using single protein species. Based on the results obtained from research funded under NASA grant NAG-8-1080.S, we conclude that binary separations are not possible using purely oscillatory flow modes because of end effects associated with the cos((omega)t) mode. Our research shows, however, that a combination of cos(2(omega)t) and steady flow should lead to efficient separation free of end effects. This possibility is currently under investigation.

  5. 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

    2011-01-01

    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 PMID:22567044

  6. Separation of Teff Eragrostis tef (Zucc.) Trotter seed proteins by capillary electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Teff (Eragrostis tef (Zucc.) Trotter) is an important food grain in Ethiopia where it is used in the preparation of the tradional flatbread injera. Teff is also used in celiac-safe food products due to its gluten-free status. Limited research has been reported on protein properties of this interesti...

  7. Identification of cultivars of Stylosanthes capitata Vog. by polyacrylamide gel electrophoresis of seed proteins

    Microsoft Academic Search

    A. Hussain; H. Ramirez; W. Bushuk; W. M. Roca

    1988-01-01

    A method was developed for identification of cultivars of the pasture legume, Stylosanthes capitata Vog., using electrophoretic patterns of seed proteins in polyacrylamide gels as the genotypic markers. The method can be used for accurate identification of cultivars in germplasm banks, in selecting parents for development of new varieties, and in registering new cultivars for proprietary purposes.

  8. Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two bacterial whole cell protein

    E-print Network

    Pal, Debnath

    Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two by two-dimensional gel electrophoresis. J. Commun. Dis. 38 255­262 Blokpoel MC, Smeulders MJ, Hubbard JA of IEF-program for 7 cm and 17 cm IPG strips. #12;Supplementary figure 6. 2DE gel images of E. coli WCPE

  9. Quantitative proteomics to study mitogen-activated protein kinases.

    PubMed

    Blagoev, Blagoy; Mann, Matthias

    2006-11-01

    In the last several years, the impact of mass spectrometry (MS)-based proteomics on cell signaling research has increased dramatically. This development has been driven both by better instrumentation and by the progression of proteomics from mainly qualitative measurements towards quantitative analyses. In this regard, Stable Isotope Labeling by Amino acids in Cell culture (SILAC) has established itself as one of the most popular and useful quantitative proteomic methodologies to study signaling networks. SILAC relies on the metabolic incorporation of non-radioactive heavy isotopes in the whole proteome of desired cell line, making all proteins from these cells easily distinguishable in the mass spectrometers from the proteins originating from control cells. The procedure does not involve any chemical derivatization steps and, importantly, allows mixing of the two cell populations for combined additional sample manipulation, thus leading to highly reliable results with minimal errors. In this chapter, we describe in detail the SILAC labeling procedure and explain how to design SILAC experiments to examine the level and duration of phosphorylation of endogenous MAP kinases and their substrates in cell culture systems. PMID:17071406

  10. Sub-visible particle quantitation in protein therapeutics.

    PubMed

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

    2009-10-01

    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

  11. Evaluation of methods for the quantitation of cysteines in proteins.

    PubMed

    Wright, S K; Viola, R E

    1998-12-01

    Several methods for the quantitation of cysteines in proteins have been evaluated and compared. Titration of protein sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) under carefully controlled conditions has extended the detection limits of this method with high accuracy and reproducibility. Results are reported for a variety of enzymes containing a range of total cysteines with different degrees of solvent accessibility and reactivity. A papain amplification assay has also been examined, in which reactivation of the disulfide-blocked active site cysteine of papain can be achieved by a coupled reaction with protein sulfhydryl groups. Detection of sulfhydryls by this amplification assay can be extended, by increasing the enzyme assay times, to achieve over a 40-fold increase in sensitivity over the improved DTNB titration method. Alternatively, titration of enzyme cysteinyl residues with either bromobimane or a maleimide derivative of naphthopyranones has the advantage that a fluorescent product results upon modification of the sulfhydryl group. Reaction of bromobimane with several different enzymes results in nonspecific background fluorescence that limits the detection range of this method unless the products are separated. In contrast, low background fluorescence and high quantum yields with maleimide naphthopyranoses has allowed detection of protein cysteinyl residues with very high sensitivities. PMID:9866701

  12. Capillary electrophoresis coupled with electrochemiluminescence for determination of atomoxetine hydrochloride and the study on its interactions with three proteins.

    PubMed

    Zeng, Hua-Jin; Yang, Ran; Zhang, Ying; Li, Jian-Jun; Qu, Ling-Bo

    2015-03-01

    A simple, rapid and sensitive method for the determination of atomoxetine hydrochloride (AH) by capillary electrophoresis with electrochemiluminescence detection (CE-ECL) using tris(2,2'-bipyridyl) ruthenium (II) was developed. Under optimized conditions, the determinations of AH in capsules and rat plasmas and the study on its interactions with three plasma proteins, including bovine serum albumin, cytochrome c and myoglobin were performed successfully. Relative to some previous studies, in this paper the methodologies for the determination of AH in aqueous solution and spiked plasma systems were established, respectively. By comparing the difference between the two work curves of two systems, the matrix effect in plasma samples on the determination of AH by the CE-ECL method was discussed in detail. The results indicated that the effect of the matrix in plasma samples should not be ignored even if no obvious interference was found in the electropherograms and the establishment of method validation in complex samples by the CE-ECL method was necessary. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25044877

  13. Quaternized cellulose-supported gold nanoparticles as capillary coatings to enhance protein separation by capillary electrophoresis.

    PubMed

    You, Jun; Zhao, Lingguo; Wang, Gongwei; Zhou, Haitao; Zhou, Jinping; Zhang, Lina

    2014-05-23

    Gold nanoparticles (Au NPs) were synthesized and stabilized by using water-soluble quaternized cellulose (QC) as support matrix through a straightforward and environmentally friendly aqueous-phase approach. The structure and morphology of QC-supported Au NPs (QC-Au NPs) were investigated systematically by UV-visible, FT-IR, x-ray diffraction and TEM measurement. The Au NPs with mean diameter of about 7nm were shown to efficiently redisperse in water due to the strong interaction between QC and Au NPs, and the solutions were quite stable after storage for nearly 4 months at room temperature. QC-Au NPs were subsequently used as novel physically adsorbed coatings for protein separation by CE. The separation performance was significantly improved in the capillary coated by QC-Au NPs compared with that of the uncoated capillary or QC coated capillary. A small quantity of Au NPs (Au content of 4.6%) was adequate for the obvious improvement of coating ability. The theoretical plate number of lysozyme in QC-Au1 NPs coated capillary was 2.9 times as much as that in QC coated capillary. We have demonstrated the separation of six model proteins with RSD of migration time less than 2.79% and RSD of peak area less than 4.81%. Furthermore, QC-Au NPs was applied to the analysis of closely related proteins and biological samples. With simplicity, high resolution and reproducibility, the proposed method shows potential for applications in proteomics and clinical diagnosis. PMID:24745845

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

    PubMed Central

    Rupp, R A; Sippel, A E

    1987-01-01

    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 PMID:3122180

  15. Capillary Electrophoresis for the Analysis of Biopolymers

    E-print Network

    Krylov, Sergey

    becoming available for some parts of proteomic research, but manually intensive slab-gel electrophoresis instruments will displace cumbersome slab-gel electrophoresis for protein analysis. We also believeCapillary Electrophoresis for the Analysis of Biopolymers Sergey N. Krylov and Norman J. Dovichi

  16. [Identification of the apomixis in Poa pratensis L. using SDS-electrophoresis of endosperm reserve proteins].

    PubMed

    Agafonov, A V; Sukhareva, N B; Baturin, S O; Struzhkova, O A

    2004-01-01

    To determine the characteristics of variability of seed reproduction in the Kentucky bluegrass (Poa pratensis), individual seed variability with respect to the composition of endospermal reserve proteins was studied. Comparative analysis of caryopses obtained by self-fertilization and free fertilization of plants I1 (no. 2-4) and I2 (no. 2-4-7) of the wild-type specimen Murmanskii-95 was performed using SDS-PAGE. Using a cytoembryological express method, we demonstrated that facultative stimulation-autonomous apomeiotic apomixis, along with the formation of meiotic megasporocytes, is characteristic of the Kentucky bluegrass. This method made it possible to determine the consequences of meiotic processes in the maternal plant and to reveal the hybrid nature of seed endosperm. PMID:15049068

  17. A Single-Sample Method for Determination of Carbohydrate and Protein Contents Glycoprotein Bands Separated by Sodium Dodecyl Sulfate– Polyacrylamide Gel Electrophoresis

    Microsoft Academic Search

    Ewa Zdebska; Jerzy Ko?cielak

    1999-01-01

    A method is described for determination of carbohydrate and protein contents of glycoproteins separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were stained, and appropriate pieces of PVDF membranes were excised, destained, and subjected to sequential hydrolysis with 0.2 M trifluoroacetic acid (TFA) for 1 h at 80°C, then with 2 M

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

    E-print Network

    Hsu, Wen-Lian

    -Q is evaluated with a mixture of 10 standard proteins and human Jurkat T cells. The results are consistent dimension in multiplexed quantitation for relative protein expression measurement in different cell states's9 . In the search for disease-associated protein determinants, quantitation remains a vital

  19. Simulating Electrophoresis.

    ERIC Educational Resources Information Center

    Moertel, Cheryl; Frutiger, Bruce

    1996-01-01

    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)

  20. Gel Electrophoresis

    NSDL National Science Digital Library

    Julie Yu

    2007-01-01

    In this activity, learners simulate the process of DNA fingerprinting by using electricity to separate colored dyes. Learners use simple materials to assemble a comb (electrophoresis chamber) to hold the samples, make a 0.2% sodium bicarbonate buffer and 1% gel solution, connect a high voltage power supply, and prepare 5 different samples. Then learners test their model and observe each sample.

  1. A new quantitative optical biosensor for protein characterisation.

    PubMed

    Cross, Graham H; Reeves, Andrew A; Brand, Stuart; Popplewell, Jonathan F; Peel, Louise L; Swann, Marcus J; Freeman, Neville J

    2003-12-15

    A new optical biosensor is described based on a dual waveguide interferometric technique. By addressing the waveguide structure with alternate polarisations the optogeometrical properties (density and thickness) of adsorbed protein layers at the sensor (solid)-liquid interface have been determined. Differences in the waveguide mode dispersion between the transverse electric (TE) and transverse magnetic (TM) modes allow unique solutions for adlayer thickness and refractive index to be determined at all stages during the formation process. The technique has been verified using standard protein systems and by comparing the data with published work using X-ray crystallography and neutron reflection techniques. The data obtained was found to be in excellent agreement with previously reported X-ray experiments given that typical film thicknesses for streptavidin layers were in the range 5.5-6.5 nm compared with the short axis crystal structure of between 4.8 and 5.6 nm. The precision of the measurements taken was of the order of 40 pm with respect to adsorbed adlayer thicknesses. This biosensor approach provides measurements of both thickness and density of adlayers to a high precision, simultaneously and in real time enabling detail of the structure and function of proteins to be elucidated. From such data it is possible to obtain information on the orientation, distortion and efficiency of immobilisation procedures as well as the interaction event of interest. The technique is expected to find utility with those interested in protein structure and function. This is an area of growing importance within the life sciences as the demand for quantitative analytical techniques increases with the growth in "proteomics". PMID:14615097

  2. New methods based on capillary electrophoresis for in vitro evaluation of protein tau phosphorylation by glycogen synthase kinase 3-?.

    PubMed

    Nehmé, Hala; Chantepie, Sandrine; Defert, Justine; Morin, Philippe; Papy-Garcia, Dulce; Nehmé, Reine

    2015-04-01

    The hyperphosphorylation of tau protein is associated with the development of the neuronal pathology of Alzheimer's disease. As most conventional methods study only particular phosphorylation sites of tau, it is necessary to develop a simple and reliable assay to determine the phosphorylation of tau at multiple sites. Capillary electrophoresis (CE)-based enzymatic assays are not yet used to monitor tau phosphorylation. The present work aims to develop CE-based assays to evaluate tau phosphorylation by the glycogen synthase kinase 3-? (GSK3?). A novel pre-capillary CE assay was first developed. An in-capillary CE-based enzymatic assay was also used since this approach is known to be time- and cost- effective. The enzymatic reaction was monitored by quantifying the product adenosine 5'- diphosphate (ADP). The influence of two classes of glycosaminoglycan (GAG), namely heparin and heparan sulfate, on the phosphorylation reaction was also assessed. Results obtained by both CE approaches were comparable and in excellent agreement with those reported in the literature using conventional radiometric and immunoblotting methods. In fact, CE results confirmed the inductory effect of the sulfated sugars heparin and heparan sulfate on tau hyperphosphorylation, probably because of the exposition of new sites phosphorylatable by GSK3?. This study shows that simple (no-labeling), rapid (less than 30 min per assay), and eco-friendly (no-radioactivity) CE-based kinase assays can give insight into the abnormal phosphorylation of tau. They can be extended to screen different modulators of tau phosphorylation to highlight their function and to develop effective drugs for neurodegenerative disease treatments. PMID:25711986

  3. Quantitative analysis of flagellar proteins in Drosophila sperm tails.

    PubMed

    Mendes Maia, Teresa; Paul-Gilloteaux, Perrine; Basto, Renata

    2015-01-01

    The cilium has a well-defined structure, which can still accommodate some morphological and molecular composition diversity to suit the functional requirements of different cell types. The sperm flagellum of the fruit fly Drosophila melanogaster appears as a good model to study the genetic regulation of axoneme assembly and motility, due to the wealth of genetic tools publically available for this organism. In addition, the fruit fly's sperm flagellum displays quite a long axoneme (?1.8mm), which may facilitate both histological and biochemical analyses. Here, we present a protocol for imaging and quantitatively analyze proteins, which associate with the fly differentiating, and mature sperm flagella. We will use as an example the quantification of tubulin polyglycylation in wild-type testes and in Bug22 mutant testes, which present defects in the deposition of this posttranslational modification. During sperm biogenesis, flagella appear tightly bundled, which makes it more challenging to get accurate measurements of protein levels from immunostained specimens. The method we present is based on the use of a novel semiautomated, macro installed in the image processing software ImageJ. It allows to measure fluorescence levels in closely associated sperm tails, through an exact distinction between positive and background signals, and provides background-corrected pixel intensity values that can directly be used for data analysis. PMID:25837396

  4. The development of simple and sensitive small-molecule fluorescent probes for the detection of serum proteins after native polyacrylamide gel electrophoresis.

    PubMed

    Wang, Fangfang; Huang, Lingyun; Na, Na; He, Dacheng; Sun, Dezhi; Ouyang, Jin

    2012-05-21

    In this paper, a simple and sensitive small-molecule fluorescent probe, 2,5-dihydroxy-4'-dimethylaminochalcone (DHDMAC), was designed and synthesized for the detection of human serum proteins via hydrophobic interactions after polyacrylamide gel electrophoresis (PAGE). This probe produced lower fluorescence emission in the absence of proteins, and the emission intensity was significantly increased after the interaction with serum proteins. To demonstrate the imaging performance of this probe as a fluorescent dye, a series of experiments was conducted that included sensitivity comparison and 2D-PAGE. The results indicated that the sensitivity of DHDMAC staining is comparable to that of the most widely used fluorescent dye, SYPRO Ruby, and more protein spots (including thyroxine-binding globulin, angiotensinogen, afamin, zinc-?-2-glycoprotein and ?-1-antichymotrypsin) were detected after 2D-PAGE. Therefore, DHDMAC is a good protein reporter due to its fast staining procedure, low detection limits and high resolution. PMID:22475746

  5. Supported molecular matrix electrophoresis: a new membrane electrophoresis for characterizing glycoproteins.

    PubMed

    Matsuno, Yu-ki; Kameyama, Akihiko

    2014-01-01

    Protein blotting is often used for identification and characterization of proteins on a membrane to which proteins separated by gel electrophoresis are transferred. The transferring process is sometimes problematic, in particular, for mucins and proteoglycans. Here, we describe a novel membrane electrophoresis technique, termed supported molecular matrix electrophoresis (SMME), in which a porous polyvinylidene difluoride (PVDF) membrane filter is used as the separation support. Proteins separated by this method can be immunoblotted without any transferring procedures. PMID:25117247

  6. Electrophoresis experiments in microgravity

    NASA Technical Reports Server (NTRS)

    Snyder, Robert S.; Rhodes, Percy H.

    1991-01-01

    The use of the microgravity environment to separate and purify biological cells and proteins has been a major activity since the beginning of the NASA Microgravity Science and Applications program. Purified populations of cells are needed for research, transplantation and analysis of specific cell constituents. Protein purification is a necessary step in research areas such as genetic engineering where the new protein has to be separated from the variety of other proteins synthesized from the microorganism. Sufficient data are available from the results of past electrophoresis experiments in space to show that these experiments were designed with incomplete knowledge of the fluid dynamics of the process including electrohydrodynamics. However, electrophoresis is still an important separation tool in the laboratory and thermal convection does limit its performance. Thus, there is a justification for electrophoresis but the emphasis of future space experiments must be directed toward basic research with model experiments to understand the microgravity environment and fluid analysis to test the basic principles of the process.

  7. Beyond hairballs: the use of quantitative mass spectrometry data to understand protein-protein interactions

    PubMed Central

    Gingras, Anne-Claude; Raught, Brian

    2012-01-01

    The past 10 years have witnessed a dramatic proliferation in the availability of protein interaction data. However, for interaction mapping based on affinity purification coupled with mass spectrometry (AP-MS), there is a wealth of information present in the datasets that often goes unrecorded in public repositories, and as such remains largely unexplored. Further, how this type of data is represented and used by bioinformaticians has not been well established. Here, we point out some common mistakes in how AP-MS data are handled, and describe how protein complex organization and interaction dynamics can be inferred using quantitative AP-MS approaches. PMID:22710165

  8. ProteinAnalysis Electrophoresis

    E-print Network

    Lebendiker, Mario

    , & Sterols PAGE IEF (Acrylamide) Agarose Gel PVDF Membrane Cellulose Acetate Membrane Nitrocellulose Membrane Nylon Membrane MALDI-MS High Sensitivity (10 ng) Higher Sensitivity (1 ng) Highest Sensitivity (0.1 ng

  9. A Prototype Two-Dimensional Capillary Electrophoresis System Fabricated in

    E-print Network

    Prentiss, Mara

    gel electrophoresis in a capillary format is presented. In this method, separation in the first electrophoresis.6,7 One-dimensional (1D) gel electrophoresis (in the form of SDS-PAGE) is still used for separations of proteins, but 1D gels are increasingly being replaced by capillary electrophoresis.8

  10. On-line preconcentration of sodium dodecyl sulfate-protein complexes using electrokinetic supercharging method with a prefilled water plug in capillary sieving electrophoresis.

    PubMed

    Liu, Jing; Kang, Mingchao; Liu, Zhen

    2011-09-01

    An electrokinetic supercharging (EKS) method with a prefilled water plug at the head column of capillary was developed for on-line preconcentration of sodium dodecyl sulfate (SDS)-protein complexes in capillary sieving electrophoresis (CSE). Conventional EKS is a combination of electrokinetic injection with transient isotachophoresis (tr-ITP). The capillary is first filled with background electrolyte, then an appropriate amount of a leading electrolyte is filled and electro-injection is carried out for certain duration. After that, terminating electrolyte is filled, and tr-ITP is subsequently initiated, followed by capillary electrophoresis (CE) separation. In this work, the performance of EKS was evaluated by integrating multiple sub-methods step by step, and a water plug containing polymer was introduced before electrokinetic injection in order to further improve the concentration effect. The positive effects of the sub-methods were verified, including molecular sieving effect of polymer, field enhanced sample injection (FESI) with and without a water plug, and transient isotachophoretic electrophoresis-based FESI. It was observed that analyte discrimination usually encountered in conventional electrokinetic injection was eliminated due to the similar charge to mass ratios of SDS-protein complexes. Based on these results, a hybrid on-line preconcentration method, EKS with injecting a water plug containing polymer before sample electrokinetic injection, was proposed and used to indiscriminately preconcentrate SDS-protein complexes, which provided a sensitivity enhancement factor of more than 1000. It was very suitable for the analysis of low-abundance proteins, providing the information of their molecular mass. PMID:22233073

  11. In-line separation by capillary electrophoresis prior to analysis by top-down mass spectrometry enables sensitive characterization of protein complexes.

    PubMed

    Han, Xuemei; Wang, Yueju; Aslanian, Aaron; Fonslow, Bryan; Graczyk, Beth; Davis, Trisha N; Yates, John R

    2014-12-01

    Intact protein analysis via top-down mass spectrometry (MS) provides a bird's eye view over the protein complexes and complex protein mixtures with the unique capability of characterizing protein variants, splice isoforms, and combinatorial post-translational modifications (PTMs). Here we applied capillary electrophoresis (CE) through a sheathless CE-electrospray ionization interface coupled to an LTQ Velos Orbitrap Elite mass spectrometer to analyze the Dam1 complex from Saccharomyces cerevisiae. We achieved a 100-fold increase in sensitivity compared to a reversed-phase liquid chromatography coupled MS analysis of recombinant Dam1 complex with a total loading of 2.5 ng (12 amol). N-terminal processing forms of individual subunits of the Dam1 complex were observed as well as their phosphorylation stoichiometry upon Mps1p kinase treatment. PMID:25382489

  12. Copper(II)-Alizarin Red S complex as an efficient chemiluminescent probe for the detection of human serum proteins after polyacrylamide gel electrophoresis.

    PubMed

    Wang, Zhenzhen; Liu, Xia; Baeyens, Willy R G; Delanghe, Joris R; Ouyang, Jin

    2008-12-01

    A novel chemiluminescent probe, copper(II)-Alizarin Red S (ARS) complex, for the detection of human serum proteins after polyacrylamide gel electrophoresis (PAGE) is described. The detection is based on the binding of the copper(II)-ARS complex to proteins and the catalytic activity of copper(II) in the luminol-hydrogen peroxide chemiluminescence (CL) system. Various proteins are directly detected in polyacrylamide gels, avoiding tedious transferring procedures. In the present study, the possible reaction mechanism and sensitivity evaluation are analyzed. The experimental conditions such as solution concentration, complex ratio, and washing reagents are likewise optimized. The proposed method offers simple, fast, and sensitive detection of serum proteins. As a novel chemiluminescent detection method, it shows significant analytical potential in biochemistry. PMID:19367697

  13. Quantitative analysis of pheromone-binding protein specificity

    PubMed Central

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

    2012-01-01

    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 (OBPs), using ?-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. 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 pheromone receptor model proposed by Laughlin et al. (Cell 133: 1255–65, 2008) are discussed. PMID:23121132

  14. Protein alterations in infiltrating ductal carcinomas of the breast as detected by nonequilibrium pH gradient electrophoresis and mass spectrometry.

    PubMed

    Kabbage, Maria; Chahed, Karim; Hamrita, Bechr; Guillier, Christelle Lemaitre; Trimeche, Mounir; Remadi, Sami; Hoebeke, Johan; Chouchane, Lotfi

    2008-01-01

    Improvement of breast-cancer detection through the identification of potential cancer biomarkers is considered as a promising strategy for effective assessment of the disease. The current study has used nonequilibrium pH gradient electrophoresis with subsequent analysis by mass spectrometry to identify protein alterations in invasive ductal carcinomas of the breast from Tunisian women. We have identified multiple protein alterations in tumor tissues that were picked, processed, and unambiguously assigned identities by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The proteins identified span a wide range of functions and are believed to have potential clinical applications as cancer biomarkers. They include glycolytic enzymes, molecular chaperones, cytoskeletal-related proteins, antioxydant enzymes, and immunologic related proteins. Among these proteins, enolase 1, phosphoglycerate kinase 1, deoxyhemoglobin, Mn-superoxyde dismutase, alpha-B-crystallin, HSP27, Raf kinase inhibitor protein, heterogeneous nuclear ribonucleoprotein A2/B1, cofilin 1, and peptidylprolyl isomerase A were overexpressed in tumors compared with normal tissues. In contrast, the IGHG1 protein, the complement C3 component C3c, which are two newly identified protein markers, were downregulated in IDCA tissues. PMID:18401453

  15. Protein Alterations in Infiltrating Ductal Carcinomas of the Breast as Detected by Nonequilibrium pH Gradient Electrophoresis and Mass Spectrometry

    PubMed Central

    Kabbage, Maria; Chahed, Karim; Hamrita, Bechr; Guillier, Christelle Lemaitre; Trimeche, Mounir; Remadi, Sami; Hoebeke, Johan; Chouchane, Lotfi

    2008-01-01

    Improvement of breast-cancer detection through the identification of potential cancer biomarkers is considered as a promising strategy for effective assessment of the disease. The current study has used nonequilibrium pH gradient electrophoresis with subsequent analysis by mass spectrometry to identify protein alterations in invasive ductal carcinomas of the breast from Tunisian women. We have identified multiple protein alterations in tumor tissues that were picked, processed, and unambiguously assigned identities by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The proteins identified span a wide range of functions and are believed to have potential clinical applications as cancer biomarkers. They include glycolytic enzymes, molecular chaperones, cytoskeletal-related proteins, antioxydant enzymes, and immunologic related proteins. Among these proteins, enolase 1, phosphoglycerate kinase 1, deoxyhemoglobin, Mn-superoxyde dismutase, ?-B-crystallin, HSP27, Raf kinase inhibitor protein, heterogeneous nuclear ribonucleoprotein A2/B1, cofilin 1, and peptidylprolyl isomerase A were overexpressed in tumors compared with normal tissues. In contrast, the IGHG1 protein, the complement C3 component C3c, which are two newly identified protein markers, were downregulated in IDCA tissues. PMID:18401453

  16. Fluorescence detection for gel and capillary electrophoresis

    SciTech Connect

    Hogan, B.

    1992-07-21

    First, an indirect fluorescence detection system for the separation of proteins via gel electrophoresis. Quantities as low as 50 nanograms of bovine serum albumin and soybean trypsin inhibitor are separated and detected visually without the need for staining of the analytes. This is very similar to levels of protein commonly separated with gel electrophoresis.

  17. Identification of increased amounts of eppin protein complex components in sperm cells of diabetic and obese individuals by difference gel electrophoresis.

    PubMed

    Paasch, Uwe; Heidenreich, Falk; Pursche, Theresia; Kuhlisch, Eberhard; Kettner, Karina; Grunewald, Sonja; Kratzsch, Jürgen; Dittmar, Gunnar; Glander, Hans-Jürgen; Hoflack, Bernard; Kriegel, Thomas M

    2011-08-01

    Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ? 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, ?-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the three groups of pathological sperm proteomes reflect a disease-associated enhanced formation of predominantly proteolytically modified forms of three eppin protein complex components, possibly as a response to enduring hyperglycemia and enhanced oxidative stress. PMID:21525168

  18. Identification of Increased Amounts of Eppin Protein Complex Components in Sperm Cells of Diabetic and Obese Individuals by Difference Gel Electrophoresis*

    PubMed Central

    Paasch, Uwe; Heidenreich, Falk; Pursche, Theresia; Kuhlisch, Eberhard; Kettner, Karina; Grunewald, Sonja; Kratzsch, Jürgen; Dittmar, Gunnar; Glander, Hans-Jürgen; Hoflack, Bernard; Kriegel, Thomas M.

    2011-01-01

    Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ? 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, ?-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the three groups of pathological sperm proteomes reflect a disease-associated enhanced formation of predominantly proteolytically modified forms of three eppin protein complex components, possibly as a response to enduring hyperglycemia and enhanced oxidative stress. PMID:21525168

  19. A web-tool for visualizing quantitative protein-protein interaction data.

    PubMed

    Knight, James D R; Liu, Guomin; Zhang, Jian Ping; Pasculescu, Adrian; Choi, Hyungwon; Gingras, Anne-Claude

    2015-04-01

    Quantitative interaction proteomics data can be a challenge to efficiently analyze and subsequently present to an audience in a simple and easy to understand format that still conveys sufficient levels of information. Here we present freely accessible and open-source web tools for displaying multiple parameters from quantitative protein-protein interaction data sets in a visually intuitive format. Given a set of "bait" proteins with detected "prey" interactions, dot plots can be generated to display absolute spectral counts for the preys, relative spectral counts between baits and confidence levels for the interactions (e.g. as determined by SAINTexpress). Additional tools are available for displaying fold change results between numerous baits with their associated confidence level (e.g. resulting from intensity measurements) and pairwise bait analyses displaying spectral counts, confidence score and fold change differences in a scatter plot format. These tools make it easy for the user to identify important interaction changes, interpret their data, and present this information to others in an intuitive way. PMID:25422071

  20. Comparison of liver mitochondrial proteins derived from newborn cloned calves and from cloned adult cattle by two-dimensional differential gel electrophoresis.

    PubMed

    Takeda, Kumiko; Tasai, Mariko; Akagi, Satoshi; Watanabe, Shinya; Oe, Mika; Chikuni, Koichi; Ohnishi-Kameyama, Mayumi; Hanada, Hirofumi; Nakamura, Yoshiaki; Tagami, Takahiro; Nirasawa, Keijiro

    2011-04-01

    Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for such a developmental deficiency. However, detailed knowledge of protein expression during somatic cell nuclear transfer (SCNT) in cattle is lacking. In the present study, variations in mitochondrial protein levels between SCNT-derived and control cattle, and from calves derived by artificial insemination were investigated. Mitochondrial fractions were prepared from frozen liver samples and subjected to two-dimensional (2-D) fluorescence differential gel electrophoresis (DIGE) using CyDye™ dyes. Protein expression changes were confirmed with a volume ratio greater than 2.0 (P?proteins for SCNT cattle (n?=?4) and seven proteins for SCNT calves (n?=?6) compared to controls (P?protein patterning was observed among SCNT animals even if animals were generated from the same donor cell source. No differences were detected in two of the SCNT cattle. Moreover, there was no novel protein identified in any of the SCNT cattle or calves. In conclusion, variation in mitochondrial protein expression concentrations was observed in non-viable, neonatal SCNT calves and among SCNT individuals. This result implicates mitochondrial-related gene expression in early developmental loss of SCNT embryos. Comparative proteomic analysis represents an important tool for further studies on SCNT animals. PMID:21387454

  1. Two-dimensional electrophoresis reveals differential protein expression in high- and low-secreting variants of the rat basophilic leukaemia cell line.

    PubMed

    Carroll, K; Ray, K; Helm, B; Carey, E

    2000-07-01

    The aim of this investigation was the identification of cellular proteins that confer a high secretory phenotype on subclones of the rat basophilic leukaemia (RBL) cell line as a model of mast cell regulated degranulation. Following protein separation by two-dimensional (2-D) electrophoresis and silver staining, more than 2000 polypeptide "spots" were resolved reproducibly. Higher sample loads and Coomassie blue staining facilitated the identification by delayed extraction-matrix-assisted laser desorption/ionization (DE-MALDI) mass spectrometry of several polypeptides that were differentially expressed in the high- and low-secreting clones. Several proteins were identified whose expression could contribute to the difference in secretory phenotype. Furthermore, silver-stained 2-D gel patterns suggested differential expression of proteins in the 20-25 kDa and the pI 4.5-7.5 range, characteristic of small guanosine 5'-triphosphate (GTP)-binding proteins. By a combination of "GTP overlay" and immunoblotting, we were able to demonstrate differential expression of small GTP binding-proteins, including Rab3 proteins, in high-and low-secreting clones. The sensitivity of this complementary approach facilitated the detection of some GTP binding and Rab3 proteins, whose expression was not evident in silver-stained 2-D gels. PMID:10939461

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

    SciTech Connect

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

    2005-07-14

    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.

  3. Two-dimensional gel electrophoresis analysis of the leiomyoma interstitial fluid reveals altered protein expression with a possible involvement in pathogenesis.

    PubMed

    Ura, Blendi; Scrimin, Federica; Zanconati, Fabrizio; Arrigoni, Giorgio; Monasta, Lorenzo; Romano, Andrea; Banco, Rubina; Zweyer, Marina; Milani, Daniela; Ricci, Giuseppe

    2015-05-01

    Uterine leiomyoma is the most common smooth benign neoplasm. In the present study, we analyzed the global interstitial fluid (IF) profile of leiomyoma vs. normal myometrium to identify protein dysregulation involved in leiomyoma pathogenesis. Two-dimensional gel electrophoresis and mass spectrometry were used to generate and compare the global interstitial fluid profiles of the leiomyoma and of the normal tissue. Two proteins were validated by immunohistochemistry. By comparing the interstitial fluid profile of the leiomyoma with that of the normal myometrium, the levels of seven proteins were found to be significantly different: four structural organization proteins (desmin, prelamin-A/C, transgelin and ?-actinin-1), an inflammatory response (?1-antitrypsin), a response to oxidative stress (peroxiredoxin-2), and a folding protein (heat shock 70 kDa protein 1A/1B). Desmin, ?1-antitrypsin and peroxiredoxin-2 were upregulated in the leiomyoma, whereas heat shock 70 kDa protein 1A/1B, ?-actinin-1, prelamin-A/C and transgelin were downregulated. Desmin and ?1-antitrypsin were further validated by immunohistochemistry. By identifying proteins with altered expression levels compared to the myometrium from several pathways of the leiomyoma pathogenesis, we found the leiomyoma interstitial fluid to have a characteristic proteomic profile. A better appreciation of the pathophysiology of the disease can be useful in the development of conservative treatments that serve as viable alternatives to hysterectomy. PMID:25738828

  4. Psoriasin, one of several new proteins identified in nasal lavage fluid from allergic and non-allergic individuals using 2-dimensional gel electrophoresis and mass spectrometry

    PubMed Central

    Bryborn, Malin; Adner, Mikael; Cardell, Lars-Olaf

    2005-01-01

    Background Extravasation and luminal entry of plasma occurs continuously in the nose. This process is markedly facilitated in patients with symptomatic allergic rhinitis, resulting in an increased secretion of proteins. Identification of these proteins is an important step in the understanding of the pathological mechanisms in allergic diseases. DNA microarrays have recently made it possible to compare mRNA profiles of lavage fluids from healthy and diseased patients, whereas information on the protein level is still lacking. Methods Nasal lavage fluid was collected from 11 patients with symptomatic allergic rhinitis and 11 healthy volunteers. 2-dimensional gel electrophoresis was used to separate proteins in the lavage fluids. Protein spots were picked from the gels and identified using mass spectrometry and database search. Selected proteins were confirmed with western blot. Results 61 spots were identified, of which 21 were separate proteins. 6 of these proteins (psoriasin, galectin-3, alpha enolase, intersectin-2, Wnt-2B and hypothetical protein MGC33648) had not previously been described in nasal lavage fluids. The levels of psoriasin were markedly down-regulated in allergic individuals. Prolactin-inducible protein was also found to be down-regulated, whereas different fragments of albumin together with Ig gamma 2 chain c region, transthyretin and splice isoform 1 of Wnt-2B were up-regulated among the allergic patients. Conclusion The identification of proteins in nasal lavage fluid with 2-dimensional gelelectrophoresis in combination with mass spectrometry is a novel tool to profile protein expression in allergic rhinitis and it might prove useful in the hunt for new therapeutic targets or diagnostic markers for allergic diseases. Psoriasin is a potent chemotactic factor and its down-regulation during inflammation might be of importance for the outcome of the disease. PMID:16236163

  5. The instantaneous monitoring of polyacrylamide gels during electrophoresis.

    PubMed Central

    Elliott, A

    1976-01-01

    The advantages of being able to see protein zones in a gel during electrophoresis (and hence before staining) are pointed out, and a method is described which depends on local increments of refractive index in these zones. The use of local increments of refractive index in polyacrylamide gels for measuring protein concentrations in zones during electrophoresis is briefly considered; it is found that such increments are greater than would be expected from the amount of protein when sodium dodecyl sulphate is present. The enhancement depends on conditions and time of running. This makes quantitative estimates difficult, but the sensitivity of detection of protein zones by observations based on refractive-index changes is greatly increased by this property of sodium dodecyl sulphate. Methods are described for making optically uniform gels (both with uniform and with graded concentrations of polyacrylamide), necessary for observation of small changes in refractive index. A simple dark-field system of observation is described. Examples are given showing protein samples observed with the system during electrophoresis and compared with the same gel stained with Coomassie Blue after completion of the run. Under optimal conditions the optical method is comparable in sensitivity with staining. With the proteins of lower mol.wt. (approx. 15000), the optical method is not so sensitive, becoming less sensitive with longer running time. This loss of sensitivity is greatly decreased by using more concentrated polyacrylamide gels, and graded gels are therefore more suitable for optical observation than are uniform gels. The observation of protein zones during electrophoresis adds nothing to the time needed for making a stained gel and gives much information long before it can be obtained from the stained gel. Images PLATE 1 PLATE 2 PMID:1008832

  6. Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS).

    PubMed

    Scheler, C; Lamer, S; Pan, Z; Li, X P; Salnikow, J; Jungblut, P

    1998-05-01

    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

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

    Microsoft Academic Search

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

    1993-01-01

    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

  8. Proteins pattern alteration in AZT-treated K562 cells detected by two-dimensional gel electrophoresis and peptide mass fingerprinting

    PubMed Central

    D'Andrea, Gabriele; Lizzi, Anna R; Venditti, Sara; Di Francesco, Laura; Giorgi, Alessandra; Mignogna, Giuseppina; Oratore, Arduino; Bozzi, Argante

    2006-01-01

    In this study we report the effect of AZT on the whole protein expression profile both in the control and the AZT-treated K562 cells, evidenced by two-dimensional gel electrophoresis and peptide mass fingerprinting analysis. Two-dimensional gels computer digital image analysis showed two spots that appeared up-regulated in AZT-treated cells and one spot present only in the drug exposed samples. Upon extraction and analysis by peptide mass fingerprinting, the first two spots were identified as PDI-A3 and stathmin, while the third one was proved to be NDPK-A. Conversely, two protein spots were present only in the untreated K562 cells, and were identified as SOD1 and HSP-60, respectively. PMID:16571109

  9. Quantitative studies in effects of additives on protein aggregation

    E-print Network

    Shinde, Chetan (Chetan Ulhas)

    2007-01-01

    Rational design of protein additives has been limited by the understanding of mechanism of protein and additive interaction. In this work we have applied molecular dynamics with all atom potentials in order to study the ...

  10. A novel (18)O inverse labeling-based workflow for accurate bottom-up mass spectrometry quantification of proteins separated by gel electrophoresis.

    PubMed

    Santos, Hugo M; Glez-Peña, Daniel; Reboiro-Jato, Miguel; Fdez-Riverola, Florentino; Diniz, Mário S; Lodeiro, Carlos; Capelo-Martínez, José-Luis

    2010-10-01

    In the present work we report on a novel and fast protocol for accurate bottom-up protein quantification that overcomes the drawbacks of in-gel digestion and MALDI analysis, while maintaining their benefits. It relies on the following steps: (i) gel electrophoresis separation of proteins, (ii) fast in-gel protein digestion with trypsin, (iii) (18)O-labeling through the decoupled method, (iv) quantification through selected peptides previously chosen using the (18)O inverse labeling approach and that, finally, (v) it takes advantage of software specifically developed to select the peptides that will drive the quantification of the protein in an automated mode. We have accurately quantified the following six proteins: glycogen phosphorylase, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor, and ?-lactalbumin. As a case study we have quantified the protein vitellogenin in plasma of Cyprinus carpio exposed to high levels of estrogens. The proposed new protocol was validated against the traditional ELISA method; both were found to provide comparable results (non-parametric Mann-Whitney U-test). PMID:20882554

  11. Variation and Genomic Localization of Genes Encoding DROSOPHILA MELANOGASTER Male Accessory Gland Proteins Separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

    PubMed Central

    Whalen, Michael; Wilson, Thomas G.

    1986-01-01

    Accessory gland proteins from Drosophila melanogaster males have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into nine major bands. When individual males from 175 strains were examined, considerable polymorphism for nearly one-half of the major protein bands was seen, including null alleles for three bands. Variation was observed not only among long-established laboratory strains but also among stocks recently derived from natural populations. There was little difference in the amount of variation between P and M strains, indicating that P element mutagenesis is not a factor producing the variation. Codominant expression of variants for each of five bands was found in heterozygotes, suggesting structural gene variation and not posttranslational modification variation. Stocks carrying electrophoretic variants of four of the major proteins were used to map the presumed structural genes for these proteins; the loci were found to be dispersed on the second chromosome. Since males homozygous for variant proteins were fertile, the polymorphism seems to have little immediate effect on successful sperm transfer. We propose that a high degree of polymorphism can be tolerated because these proteins play a nutritive rather than enzymatic role in Drosophila reproduction. PMID:3095182

  12. Gel Electrophoresis on a Budget to Dye For

    NSDL National Science Digital Library

    Julie H. Yu

    2010-01-01

    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

  13. Detection of metals in proteins by means of polyacrylamide gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry: application to selenium.

    PubMed

    Chéry, Cyrille C; Günther, Detlef; Cornelis, Rita; Vanhaecke, Frank; Moens, Luc

    2003-10-01

    The capabilities of laser ablation-inductively coupled plasma-mass spectrometry for the detection of trace elements in a gel after gel electrophoresis were systematically studied. Figures of merit, such as limit of detection, linearity, and repeatability, were evaluated for various elements (Li, V, Cr, Mn, Ni, Cu, Zn, As, Se, Mo, Pd, Ag, Cd, Pt, Tl, Pb). Two ablation strategies were followed: single hole drilling, relevant for ablation of spots after two-dimensional (2-D) separations, and ablation with translation, i.e., on a line, relevant for one-dimensional (1-D) separations. This technique was applied to the detection of selenoproteins in red blood cells extracts after a 1-D separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the detection of selenium-containing proteins in yeast after 2-D electrophoresis (2-DE). The detection procedure was further improved by using the dynamic reaction cell technology, which allowed the removal of the Ar_2(+) interference and hence the use of the most abundant Se isotope, (80)Se. Reaction gases were compared (methane, carbon monoxide, ammonia, oxygen and the combination of argon (collision gas) and hydrogen (reaction gas)). In each instance, the reaction cell parameters were optimized in order to obtain the lowest detection limit for Se (as (80)Se(+), (82)Se(+) or (77)Se(+); and as (80)Se(16)O(+), (82)Se(16)O(+) or (77)Se(16)O(+) with O(2) as the reaction gas). Carbon monoxide was found to offer the best performance. The detection limit with the use of DRC and He as transport gas was 0.07 microg Se g(-1) gel with single hole drilling and 0.15 microg Se g(-1) gel for ablation with translation. PMID:14595676

  14. A novel specific heparin-binding activity of bovine folate-binding protein characterized by capillary electrophoresis.

    PubMed

    Heegaard, Niels H H; Hansen, Steen I; Holm, Jan

    2006-03-01

    Folate-binding proteins (FBPs) are ubiquitous, soluble and membrane-bound high-affinity receptors for folate, an essential nutrient involved in nucleic and amino acid metabolism. In the course of optimizing CE separation conditions for FBP purified from cow's milk we discovered a novel specific heparin-binding activity of FBP by affinity CE. Heparin is a highly sulfated glycosaminoglycan and thus prone to induce anodic migration shifts of complexing analytes. Prior complexation of FBP with folate abolished heparin binding, and thus folate competes with heparin for binding to FBP. It was estimated that heparin bound several orders of magnitude less strongly than folate with an average dissociation constant in the 1-10 microM range. In contrast to the mobility shifts induced by heparin, free and folate-bound FBP were not separated by CE. However, binding of folate induced a distinct increase in FBP-peak symmetry, and using heparin as an affinity displacer, the free FBP in equilibrium with folate-FBP complexes could readily be separated from the complexes. While the folate-FBP interaction was too strong to be characterized quantitatively because of inadequate detection limits of a UV-based detection system, it was possible to estimate the folate-FBP binding stoichiometry using this approach. The heparin interaction fractionated FBP into distinct subfractions, and the CE approach thus promises to be useful for unraveling the complex oligomerization behavior of FBP isoforms as well as for evaluating the FBP affinity for various species and analogs of glycosaminoglycans and folate. PMID:16470783

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

    Microsoft Academic Search

    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

    2004-01-01

    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

  16. A Microfluidic Platform for High-Throughput Multiplexed Protein Quantitation

    PubMed Central

    Volpetti, Francesca; Garcia-Cordero, Jose; Maerkl, Sebastian J.

    2015-01-01

    We present a high-throughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. Samples are quantitated in each unit cell by four independent MITOMI detection areas, allowing four samples to be analyzed in parallel for a total of 1,536 assays per device. We show that the device can be pre-assembled and stored for weeks at elevated temperature and we performed proof-of-concept experiments simultaneously quantitating IL-6, IL-1?, TNF-?, PSA, and GFP. Finally, we show that the platform can be used to identify functional antibody combinations by screening 64 antibody combinations requiring up to 384 unique assays per device. PMID:25680117

  17. Exposures of Sus scrofa to a TASER(®) conducted electrical weapon: no effects on 2-dimensional gel electrophoresis patterns of plasma proteins.

    PubMed

    Jauchem, James R; Cerna, Cesario Z; Lim, Tiffany Y; Seaman, Ronald L

    2014-12-01

    In an earlier study, we found significant changes in red-blood-cell, leukocyte, and platelet counts, and in red-blood-cell membrane proteins, following exposures of anesthetized pigs to a conducted electrical weapon. In the current study, we examined potential changes in plasma proteins [analyzed via two-dimensional gel electrophoresis (2-DGE)] following two 30 s exposures of anesthetized pigs (Sus scrofa) to a TASER (®) C2 conducted electrical weapon. Patterns of proteins, separated by 2-DGE, were consistent and reproducible between animals and between times of sampling. We determined that the blood plasma collection, handling, storage, and processing techniques we used are suitable for swine blood. There were no statistically significant changes in plasma proteins following the conducted-electrical-weapon exposures. Overall gel patterns of fibrinogen were similar to results of other studies of both pigs and humans (in control settings, not exposed to conducted electrical weapons). The lack of significant changes in plasma proteins may be added to the body of evidence regarding relative safety of TASER C2 device exposures. PMID:25319243

  18. Getting the Most out of Electrophoresis Units

    ERIC Educational Resources Information Center

    Mulvihill, Charlotte

    2007-01-01

    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…

  19. How Many proteins are Missed in Quantitative proteomics Based on Ms/Ms sequencing Methods?

    PubMed Central

    Mulvey, Claire; Thur, Bettina; Crawford, Mark; Godovac-Zimmermann, Jasminka

    2014-01-01

    Current bottom-up quantitative proteomics methods based on MS/MS sequencing of peptides are shown to be strongly dependent on sample preparation. Using cytosolic proteins from MCF-7 breast cancer cells, it is shown that protein pre-fractionation based on pI and MW is more effective than pre-fractionation using only MW in increasing the number of observed proteins (947 vs. 704 proteins) and the number of spectral counts per protein. Combination of MS data from the different pre-fractionation methods results in further improvements (1238 proteins). We discuss that at present the main limitation on quantitation by MS/MS sequencing is not MS sensitivity and protein abundance, but rather extensive peptide overlap and limited MS/MS sequencing throughput, and that this favors internally calibrated methods such as SILAC, ICAT or ITRAQ over spectral counting methods in attempts to drastically improve proteome coverage of biological samples.

  20. Quantitation of GFP-fusion proteins in single living cells

    Microsoft Academic Search

    Miroslav Dundr; James G. McNally; Jean Cohen; Tom Misteli

    2002-01-01

    The green fluorescent protein (GFP) has revolutionized cell biology. The ability to observe genetically encoded fluorescently tagged fusion proteins in intact cells has made virtually any biological process amenable to investigation in living cells. However, most in vivo imaging studies are qualitative and little information about the number of fluorescently labeled molecules observed in a cell or a cellular structure

  1. Electrophoresis. [in microgravity environment

    NASA Technical Reports Server (NTRS)

    Bier, M.

    1977-01-01

    Ground-based techniques for electrophoresis take account of the need either to circumvent the effects of gravity to prevent convection, or to use gravity for fluid stabilization through artificial density gradients. The microgravity environments of orbiting spacecraft provides a new alternative for electrophoresis by avoiding the need for either of these two approaches. The paper presents some theoretical considerations concerning electrophoresis, examines certain experimental techniques (zone and high density gel electrophoresis, isoelectric focusing and isotachophoresis), and examines the electrophoresis of living cells.

  2. Effects of haemolysis, lipaemia, bilirubinaemia and fibrinogen on protein electropherogram of canine samples analysed by capillary zone electrophoresis.

    PubMed

    Martínez-Subiela, S; Tecles, F; Montes, A; Gutiérrez, C; Cerón, J J

    2002-11-01

    The possible interference of haemolysis, lipaemia, bilirubinaemia and fibrinogen on capillary zone electrophoresis of canine samples were studied. Solutions of haemoglobin, lipid and bilirubin were prepared and mixed with serum aliquots to make up samples containing different concentrations of the putative interferent substance. In addition, samples of serum and plasma were assayed to assess the influence of fibrinogen. Haemolysis and lipids produced a change in electropherogram morphology giving an interference peak located in the beta-2 region when haemoglobin was increased, and in the alpha-2 region when lipids were increased. A rise in concentration of these interferents caused an increase in the beta and alpha-2 fractions respectively, and a decrease in the other fractions. Bilirubin did not alter morphology but gave an increase in the albumin and alpha-1 and a decrease in the alpha-2 and beta-2 fractions. No differences were found between serum and plasma samples, and fibrinogen did not produce any additional peak. PMID:12505401

  3. Quantitative trait loci influencing protein and starch concentration in the Illinois Long Term Selection maize strains

    Microsoft Academic Search

    I. L. Goldman; T. R. Rocheford; J. W. Dudley

    1993-01-01

    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

  4. QUANTITATIVE DOT-IMMUNOBINDING ASSAY FOR PROTEINS USING NITROCELLULOSE MEMBRANE FILTERS

    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...

  5. Optimized Protein Extraction for Quantitative Proteomics of Yeasts

    Microsoft Academic Search

    Tobias von der Haar; Thomas Preiss

    2007-01-01

    BackgroundThe absolute quantification of intracellular protein levels is technically demanding, but has recently become more prominent because novel approaches like systems biology and metabolic control analysis require knowledge of these parameters. Current protocols for the extraction of proteins from yeast cells are likely to introduce artifacts into quantification procedures because of incomplete or selective extraction.Principal FindingsWe have developed a novel

  6. Long-Term Biological Variation of Serum Protein Electrophoresis M-Spike, Urine M-Spike, and Monoclonal Serum Free Light Chain Quantification: Implications for Monitoring Monoclonal Gammopathies

    PubMed Central

    Katzmann, Jerry A.; Snyder, Melissa R.; Rajkumar, S. Vincent; Kyle, Robert A.; Therneau, Terry M.; Benson, Joanne T.; Dispenzieri, Angela

    2013-01-01

    BACKGROUND We analyzed serial data in patients with clinically stable monoclonal gammopathy to determine the total variation of serum M-spikes [measured with serum protein electrophoresis (SPEP)], urine M-spikes [measured with urine protein electrophoresis (UPEP)], and monoclonal serum free light chain (FLC) concentrations measured with immunoassay. METHODS Patients to be studied were identified by (a) no treatment during the study interval, (b) no change in diagnosis and <5 g/L change in serum M-spike over the course of observation; (c) performance of all 3 tests (SPEP, UPEP, FLC immunoassay) in at least 3 serial samples that were obtained 9 months to 5 years apart; (d) serum M-spike ?10 g/L, urine M-spike ?200 mg/24 h, or clonal FLC ?100 mg/L. The total CV was calculated for each method. RESULTS Among the cohort of 158 patients, 90 had measurable serum M-spikes, 25 had urine M-spikes, and 52 had measurable serum FLC abnormalities. The CVs were calculated for serial SPEP M-spikes (8.1%), UPEP M-spikes (35.8%), and serum FLC concentrations (28.4%). Combining these CVs and the interassay analytical CVs, we calculated the biological CV for the serum M-spike (7.8%), urine M-spike (35.5%), and serum FLC concentration (27.8%). CONCLUSIONS The variations in urine M-spike and serum FLC measurements during patient monitoring are similar and are larger than those for serum M-spikes. In addition, in this group of stable patients, a measurable serum FLC concentration was available twice as often as a measurable urine M-spike. PMID:21980167

  7. Quantitative Proteomics Identify Novel miR-155 Target Proteins

    PubMed Central

    Lößner, Christopher; Meier, Jan; Warnken, Uwe; Rogers, Michael A.; Lichter, Peter; Pscherer, Armin; Schnölzer, Martina

    2011-01-01

    Background MicroRNAs are 22 nucleotides long non-coding RNAs and exert their function either by transcriptional or translational inhibition. Although many microRNA profiles in different tissues and disease states have already been discovered, only little is known about their target proteins. The microRNA miR-155 is deregulated in many diseases, including cancer, where it might function as an oncoMir. Methodology/Principal Findings We employed a proteomics technique called “stable isotope labelling by amino acids in cell culture” (SILAC) allowing relative quantification to reliably identify target proteins of miR-155. Using SILAC, we identified 46 putative miR-155 target proteins, some of which were previously reported. With luciferase reporter assays, CKAP5 was confirmed as a new target of miR-155. Functional annotation of miR-155 target proteins pointed to a role in cell cycle regulation. Conclusions/Significance To the best of our knowledge we have investigated for the first time miR-155 target proteins in the HEK293T cell line in large scale. In addition, by comparing our results to previously identified miR-155 target proteins in other cell lines, we provided further evidence for the cell line specificity of microRNAs. PMID:21799781

  8. Identification of novel 14-3-3? interacting proteins by quantitative immunoprecipitation combined with knockdown (QUICK).

    PubMed

    Ge, Feng; Li, Wen-Liang; Bi, Li-Jun; Tao, Sheng-Ce; Zhang, Zhi-Ping; Zhang, Xian-En

    2010-11-01

    The family of 14-3-3 proteins has emerged as critical regulators of diverse cellular responses under both physiological and pathological conditions. To gain insight into the molecular action of 14-3-3? in multiple myeloma (MM), we performed a systematic proteomic analysis of 14-3-3?-associated proteins. This analysis, recently developed by Matthias Mann, termed quantitative immunoprecipitation combined with knockdown (QUICK), integrates RNAi, SILAC, immunoprecipitation, and quantitative MS technologies. Quantitative mass spectrometry analysis allowed us to distinguish 14-3-3?-interacting proteins from background proteins, resulting in the identification of 292 proteins in total with 95 novel interactions. Three 14-3-3?-interacting proteins-BAX, HSP70, and BAG3-were further confirmed by reciprocal coimmunoprecipitations and colocalization analysis. Our results therefore not only uncover a large number of novel 14-3-3?-associated proteins that possess a variety of cellular functions, but also provide new research directions for the study of the functions of 14-3-3?. This study also demonstrated that QUICK is a useful approach to detect specific protein-protein interactions with very high confidence and may have a wide range of applications in the investigation of protein complex interaction networks. PMID:20879785

  9. USE OF QUANTITATIVE TWO-DIMENSIONAL GEL ELECTROPHORESIS TO ANALYZE CHANGES IN ALVEOLAR MACROPHAGE PROTEINS IN HUMANS EXPOSED TO OZONE

    EPA Science Inventory

    Acute exposure of humans to 0.4 ppm ozone is known to cause production of components which mediate inflammation and damage in the lung. he contribution of alveolar macrophages to this process is not well understood. n addition, ozone may cause more extensive cellular changes than...

  10. Quantitative determination of curcuminoids in Curcuma rhizomes and rapid differentiation of Curcuma domestica Val. and Curcuma xanthorrhiza Roxb. by capillary electrophoresis.

    PubMed

    Lechtenberg, Matthias; Quandt, Bettina; Nahrstedt, Adolf

    2004-01-01

    The three major curcuminoids, curcumin, demethoxycurcumin and bis-demethoxycurcumin, from Curcuma domestica Val. (Curcuma longa L.) and Curcuma xanthorrhiza Roxb. (Zingiberaceae) were fully separated and quantified in less than 5 min using a capillary zone electrophoresis method with standard fused-silica capillaries and photodiode array detection. An electrolyte solution of 20 mM phosphate, 50 mM sodium hydroxide and 14 mM beta-cyclodextrin was found to be appropriate. Quantification was performed with 3,4-dimethoxy-trans-cinnamic acid as internal standard, and the limit of detection was 0.01 mg/mL. Extraction, stabilisation during sample storage and quantification procedures were optimised and carried out with drugs and commercial curry powder from different provenances. The results were compared with the photometric method of the monograph Curcumae xanthorrhizae rhizoma of the European Pharmacopoeia. PMID:15202598

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

    PubMed Central

    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

    2008-01-01

    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

  12. Labeling of capsid proteins and genomic RNA of human rhinovirus with two different fluorescent dyes for selective detection by capillary electrophoresis.

    PubMed

    Kremser, Leopold; Petsch, Martina; Blaas, Dieter; Kenndler, Ernst

    2004-12-15

    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

  13. Quantitative Analysis of Protein-Lipid Interactions Using Tryptophan Fluorescence

    NSDL National Science Digital Library

    Catherine A. Kraft (University of Pittsburgh School of Medicine; The Department of Pharmacology and Chemical Biology REV)

    2009-12-01

    The fluorescent properties of the amino acid tryptophan make it a useful tool for fluorometric assays. Because tryptophan fluorescence is remarkably sensitive to the polarity of the environment, it can be used to determine the affinity of tryptophan-containing peptides for phospholipid vesicles of varying compositions. Here, we describe a method for using tryptophan fluorescence to determine the binding affinities of peptides derived from the proteins Raf-1 and KSR-1 to small unilamellar vesicles containing phosphatidic acid. The method can be extrapolated to measure the binding of other tryptophan-containing peptides or proteins to lipid vesicles.

  14. Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.

    PubMed

    Nakajima, Kazuki; Kinoshita, Mitsuhiro; Oda, Yasuo; Masuko, Takashi; Kaku, Hanae; Shibuya, Naoto; Kakehi, Kazuaki

    2004-09-01

    We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities. PMID:15148295

  15. 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

    González-Marrero, Ibrahim; Castañeyra-Ruiz, Leandro; González-Toledo, Juan M.; Castañeyra-Ruiz, Agustín; de Paz-Carmona, Hector; Castro, Rafael; Hernandez-Fernaud, Juan R.; Castañeyra-Perdomo, Agustín; Carmona-Calero, Emilia M.

    2013-01-01

    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. PMID:23401751

  16. Dendritic glycopolymers as dynamic and covalent coating in capillary electrophoresis: view on protein separation processes and detection of nanogram-scaled albumin in biological samples.

    PubMed

    Polikarpov, Nikita; Potolytsyna, Vera; Bessonova, Elena; Tripp, Sandra; Appelhans, Dietmar; Voit, Brigitte; Kartsova, Ludmilla

    2015-01-23

    Unique properties of dendritic polymers make them promising candidates for application as additives in various analytical methods. From this point, we investigated the potential use of maltose-modified hyperbranched poly(ethylene imine) (PEI-Mal) as a dynamically or covalently bound coating and a pseudostationary phase in capillary electrophoresis. The EOF mobilities were measured at different pH values (2.2, 8.5, and 10.2) using PEI-Mal in the background electrolyte (BGE) and desirable repeatability of the EOF with % RSD (n=50) ?3.3 was obtained. The influence of pH, polymer concentration, and density of maltose shell on the separation properties of a model mixture of four proteins (albumin, lysozyme, myoglobin, insulin) were investigated. Applying PEI-Mal as a dynamic coating, improved separation of the protein mixture with a high repeatability was achieved. Applying PEI-Mal as a covalent coating for concentrating proteins in the large volume sample stacking (LVSS) combined with the field-enhanced sample injection (FESI), up to 1320-fold enhancement of sensitivity was achieved. The detection limit of 100-500ng/ml allowed successful analysis of albumin level both in blood and urine samples without additional preconcentration. PMID:25555410

  17. A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.

    PubMed

    Bohler, Sacha; Sergeant, Kjell; Hoffmann, Lucien; Dizengremel, Pierre; Hausman, Jean-Francois; Renaut, Jenny; Jolivet, Yves

    2011-07-01

    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

  18. Mercurochrom can be used for the histochemical demonstration and microphotometric quantitation of both protein thiols and protein (mixed) disulfides.

    PubMed

    Nöhammer, G; Desoye, G

    1997-05-01

    Mercurochrom [2,7-dibromo-4-(hydroxymercuri)-fluorescein disodium salt] used for staining of protein thiols in addition binds to other groups of proteins. Experimental evidence is provided that mercurochrom bound to non-thiol groups forms a 1:1 adduct with protein (mixed) disulfides. The disulfide contents of three different types of cells determined biochemically correlated with the corresponding mean integrated optical densities determined microphotometrically after mercurochrom staining of groups other than thiols. Intracellular disulfide exchange has been studied, leading to a transformation of protein mixed disulfides to protein disulfides and an equimolar loss of protein thiols. Protein mixed disulfides were generated from protein thiols using both methyl methanethiosulfonate (MMTS) and 2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD). Loss of thiols as well as the equimolar increase of protein mixed disulfides were followed using both mercurochrom staining for thiols and for disulfides. Generation of protein mixed disulfides due to the DDD reaction was also followed by azocoupling with Fast blue B. On the basis of the observed stoichiometry between the loss of protein thiols and the quantity, increase or conversion of protein disulfides determined microphotometrically using both mercurochrom staining and DDD Fast blue B staining, we conclude that: (1) 1 mol of mercurochrom is bound per mol of protein (mixed) disulfide; and (2) the molar absorptivity of mercurochrom bound to disulfides is epsilon 520 = 34940. This study demonstrates that mercurochrom can be used for the quantitative determination of the oxidative status of protein thiols in cells. PMID:9208329

  19. Detection of seminal fluid proteins in the bed bug, Cimex lectularius, using two-dimensional gel electrophoresis and mass spectrometry

    PubMed Central

    REINHARDT, K.; WONG, C. H.; GEORGIOU, A. S.

    2008-01-01

    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. PMID:19091156

  20. Detection of seminal fluid proteins in the bed bug, Cimex lectularius, using two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Reinhardt, K; Wong, C H; Georgiou, A S

    2009-03-01

    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

  1. Quantitative Proteomic Analysis of Proteins Released by Neoplastic Prostate Epithelium

    Microsoft Academic Search

    Daniel B. Martin; David R. Gifford; Michael E. Wright; Andrew Keller; Eugene Yi; David R. Goodlett; Reudi Aebersold; Peter S. Nelson

    2004-01-01

    Prostate cancer is unusual among neoplasms in that it may be diag- nosed at a curable stage through detection of a protein in serum, the serine protease prostate-specific antigen (PSA). PSA is secreted by both normal and neoplastic prostate epithelial cells in response to androgenic hor- mones and has found widespread use in cancer screening. Because PSA screening is controversial

  2. Tau protein binds single-stranded DNA sequence specifically the proof obtained in vitro with non-equilibrium capillary electrophoresis

    E-print Network

    Krylov, Sergey

    Tau protein binds single-stranded DNA sequence specifically ­ the proof obtained in vitro with non 28 January 2005 Edited by Jesus Avila Abstract Tau is a microtubule-associated protein, which plays an important role in physiology and pathology of neurons. Tau has been recently reported to bind double

  3. Rapid Separation and Quantification of Major Caseins and Whey Proteins of Bovine Milk by Polyacrylamide Gel Electrophoresis

    Microsoft Academic Search

    K. F. Ng-Kwai-Hang; E. M. Kroeker

    1984-01-01

    A rapid polyacrylamide gel electro- phoretic method was developed for separating and quantifying major pro- teins in casein and whey protein fractions of bovine milk. For casein separation, best results were achieved by an 8% poly- acrylamide gel containing 4 M urea and a top layer of large pore sample gel; for whey protein the most_ satisfactory separation was with

  4. Altered Protein Expression of Streptococcus oralis Cultured at Low pH Revealed by Two-Dimensional Gel Electrophoresis

    Microsoft Academic Search

    JOANNA C. WILKINS; KAREN A. HOMER; DAVID BEIGHTON

    2001-01-01

    Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH

  5. Serotype classification and characterisation of the rotavirus SA11 VP6 protein using mass spectrometry and two-dimensional gel electrophoresis.

    PubMed

    Emslie, K R; Molloy, M P; Barardi, C R; Jardine, D; Wilkins, M R; Bellamy, A R; Williams, K L

    2000-05-01

    VP6, which makes up the inner capsid of rotavirus, is the major structural protein of this virus. Whilst VP6 has been sequenced at the DNA level in several rotavirus strains, there has been less effort to characterise the protein at the amino acid level. This paper reports the use of peptide mass fingerprinting and post-source decay fragmentation studies using MALDI-TOF and electrospray ionisation mass spectrometry to identify and characterise, in detail, the VP6 protein. We show that mass spectrometric analysis of VP6 peptides successfully distinguished SA11 from other rotavirus serotypes, and identify unique peptides that can be used for serotypic differentiation. For VP6 characterisation, the ExPASy FindMod tool was used to predict post-translational modifications on the protein. Analysis of trypsin and AspN digests predicted that the N-terminal methionine of VP6 was acetylated and this was confirmed using post source decay and electrospray ionisation mass spectrometry-mass spectrometry. An asparagine residue (aa107), which is followed by a glycine residue, was shown to undergo partial deamidation to aspartic acid. VP6 has two additional asparagine-glycine sequences and, in this sequence context, asparagine is known to be particularly susceptible to deamidation. Two-dimensional gel electrophoresis revealed a complex series of VP6 isoforms with an apparent molecular mass of approximately 45,000 Da and a pI ranging from 5.25 to 5.8. This pattern could partly be explained by the potential for deamidation at several sites within the protein. PMID:11793218

  6. Neurofilament Protein Levels: Quantitative Analysis in Essential Tremor Cerebellar Cortex

    PubMed Central

    Louis, Elan D.; Karen; Babij, Rachel; Cortés, Etty; Liem, Ronald K.; Vonsattel, Jean-Paul G.; Faust, Phyllis L.

    2012-01-01

    Essential tremor (ET) is among the most prevalent neurological diseases. A substantial increase in the number of Purkinje cell axonal swellings (torpedoes) has been identified in ET brains. We recently demonstrated that torpedoes in ET contain an over-accumulation of disorganized neurofilament (NF) proteins. This now raises the question whether NF protein composition and/or phosphorylation state in cerebellar tissue might differ between ET cases and controls. We used a Western blot analysis to compare the levels and phosphorylation state of NF proteins and ?-internexin in cerebellar tissue from 47 ET cases vs. 26 controls (2:1 ratio). Cases and controls did not differ with respect to the cerebellar levels of NF-light (NF-L), NF-medium (NF-M), NF-heavy (NF-H), or ?-internexin. However, SMI-31 levels (i.e., phosphorylated NF-H) and SMI-32 levels (i.e., non-phosphorylated NF-H) were significantly higher in ET cases than controls (1.28 ± 0.47 vs. 1.06 ± 0.32, p = 0.02; and 1.38 ± 0.75 vs. 1.00 ± 0.42, p = 0.006). Whether the abnormal phosphorylation state that we observed is a cause of defective axonal transport and/or function of NFs in ET is not known. NF abnormalities have been demonstrated in several neurodegenerative diseases. Regardless of whether these protein aggregates are the cause or consequence of these diseases, NF abnormalities have been shown to be an important factor in the cellular disruption observed in several neurodegenerative diseases. Therefore, further analyses of these NF abnormalities and their mechanisms are important to enhance our understanding of disease pathogenesis in ET. PMID:22561033

  7. Neurofilament protein levels: quantitative analysis in essential tremor cerebellar cortex.

    PubMed

    Louis, Elan D; Ma, Karen; Babij, Rachel; Cortés, Etty; Liem, Ronald K; Vonsattel, Jean-Paul G; Faust, Phyllis L

    2012-06-14

    Essential tremor (ET) is among the most prevalent neurological diseases. A substantial increase in the number of Purkinje cell axonal swellings (torpedoes) has been identified in ET brains. We recently demonstrated that torpedoes in ET contain an over-accumulation of disorganized neurofilament (NF) proteins. This now raises the question whether NF protein composition and/or phosphorylation state in cerebellar tissue might differ between ET cases and controls. We used a Western blot analysis to compare the levels and phosphorylation state of NF proteins and ?-internexin in cerebellar tissue from 47 ET cases versus 26 controls (2:1 ratio). Cases and controls did not differ with respect to the cerebellar levels of NF-light (NF-L), NF-medium (NF-M), NF-heavy (NF-H), or ?-internexin. However, SMI-31 levels (i.e., phosphorylated NF-H) and SMI-32 levels (i.e., non-phosphorylated NF-H) were significantly higher in ET cases than controls (1.28±0.47 vs. 1.06±0.32, p=0.02; and 1.38±0.75 vs. 1.00±0.42, p=0.006). Whether the abnormal phosphorylation state that we observed is a cause of defective axonal transport and/or function of NFs in ET is not known. NF abnormalities have been demonstrated in several neurodegenerative diseases. Regardless of whether these protein aggregates are the cause or consequence of these diseases, NF abnormalities have been shown to be an important factor in the cellular disruption observed in several neurodegenerative diseases. Therefore, further analyses of these NF abnormalities and their mechanisms are important to enhance our understanding of disease pathogenesis in ET. PMID:22561033

  8. Comprehensive multiplexed protein quantitation delineates eosinophilic and neutrophilic experimental asthma

    PubMed Central

    2014-01-01

    Background Improvements in asthma diagnosis and management require deeper understanding of the heterogeneity of the complex airway inflammation. We hypothesise that differences in the two major inflammatory phenotypes of asthma; eosinophilic and neutrophilic asthma, will be reflected in the lung protein expression profile of murine asthma models and can be delineated using proteomics of bronchoalveolar lavage (BAL). Methods BAL from mice challenged with ovalbumin (OVA/OVA) alone (standard model of asthma, here considered eosinophilic) or OVA in combination with endotoxin (OVA/LPS, model of neutrophilic asthma) was analysed using liquid chromatography coupled to high resolution mass spectrometry, and compared with steroid-treated animals and healthy controls. In addition, conventional inflammatory markers were analysed using multiplexed ELISA (Bio-Plex™ assay). Multivariate statistics was performed on integrative proteomic fingerprints using principal component analysis. Proteomic data were complemented with lung mechanics and BAL cell counts. Results Several of the analysed proteins displayed significant differences between the controls and either or both of the two models reflecting eosinophilic and neutrophilic asthma. Most of the proteins found with mass spectrometry analysis displayed a considerable increase in neutrophilic asthma compared with the other groups. Conversely, the larger number of the inflammatory markers analysed with Bio-Plex™ analysis were found to be increased in the eosinophilic model. In addition, major inflammation markers were correlated to peripheral airway closure, while commonly used asthma biomarkers only reflect central inflammation. Conclusion Our data suggest that the commercial markers we are currently relying on to diagnose asthma subtypes are not giving us comprehensive or specific enough information. The analysed protein profiles allowed to discriminate the two models and may add useful information for characterization of different asthma phenotypes. PMID:24993465

  9. 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

    Baghian, A; Shaffer, L; Storz, J

    1990-01-01

    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 PMID:1691145

  10. Kidney cell electrophoresis

    NASA Technical Reports Server (NTRS)

    Todd, P.

    1979-01-01

    A kidney cell electrophoresis technique is described in four parts: (1) the development and testing of electrophoresis solutions; (2) optimization of freezing and thawing; (3) procedures for evaluation of separated kidney cells; and (4) electrophoretic mobility characteristics of kidney cells.

  11. Kidney cell electrophoresis

    NASA Technical Reports Server (NTRS)

    Todd, P.

    1980-01-01

    The following aspects of kidney cell electrophoresis are discussed: (1) the development and testing of electrophoresis solutions; (2) optimization of freezing and thawing; (3) procedures for evaluation of separated kidney cells; and (4) electrophoretic mobility characterization of kidney cells.

  12. An Economical Electrophoresis Apparatus

    ERIC Educational Resources Information Center

    Andrews, I. M.

    1975-01-01

    Describes the production of an electrophoresis apparatus from commonly discarded articles. Outlines paper and gel electrophoresis and its application to the separation of amino acids and intestinal enzymes. (GS)

  13. Mass-balanced 1H/2H isotope dipeptide tag for simultaneous protein quantitation and identification.

    PubMed

    Seo, Jongcheol; Suh, Min-Soo; Thangadurai, T Daniel; Kim, Jinhee; Rhee, Young Ho; Yoon, Hye-Joo; Shin, Seung Koo

    2008-08-15

    Mass-balanced (1)H/(2)H isotope dipeptide tags (MBITs) are presented for simultaneous protein quantitation and identification. MBIT is derived from N-acetyl-Ala-Ala dipeptide and conjugated to primary amines of target peptides. (1)H/(2)H isotopes are encoded in the methyl groups of N-acetylated dipeptide: one tag deuterated on the N-acetyl group and another on the C-terminal alanine. MBIT-linked peptides comigrate in reversed-phase liquid chromatography without significant (1)H/(2)H isotope effects and provide 2-plex quantitation signals at 114 and 117 Th as well as peptide sequence information upon MS/MS analysis with MALDI TOF/TOF. MBIT shows good quantitation linearity in a concentration range of 20-250 fmol. The performance of MBIT on protein quantitation and identification is further tested with yeast heat-shock protein (Hsp82p) obtained from three different physiological states. MBIT using nanogram-scale samples produces the relative abundance ratios comparable to those obtained from optical imaging of microgram-scale samples visualized with SYPRO Ruby stain. The MBIT strategy is a simple and low-cost alternative for 2-plex quantitation of proteins and offers possibilities of tuning the 2-plex signal mass window by replacing the N-terminal alanine with other amino acid residues. PMID:18620426

  14. Kidney cell electrophoresis

    NASA Technical Reports Server (NTRS)

    Todd, P. W.

    1985-01-01

    Tasks were undertaken in support of two objectives. They are: (1) to carry out electrophoresis experiments on cells in microgravity; and (2) assess the feasibility of using purified kidney cells from embryonic kidney cultures as a source of important cell products. Investigations were carried out in the following areas: (1) ground based electrophoresis technology; (2) cell culture technology; (3) electrophoresis of cells; (4) urokinase assay research; (5) zero-g electrophoresis; and (6) flow cytometry.

  15. Improved protein arrays for quantitative systems analysis of the dynamics of signaling pathway interactions

    PubMed Central

    2011-01-01

    An improved version of quantitative protein array platform utilizing linear Quantum dot signaling for systematically measuring protein levels and phosphorylation states is presented. The signals are amplified linearly by a confocal laser Quantum dot scanner resulting in ~1000-fold more sensitivity than traditional Western blots, but are not linear by the enzyme-based amplification. Software is developed to facilitate the quantitative readouts of signaling network activities. Kinetics of EGFRvIII mutant signaling was analyzed to quantify cross-talks between EGFR and other signaling pathways. PMID:21917185

  16. Gel Electrophoresis and Photography

    E-print Network

    Simpson, Larry

    Gel Electrophoresis and Photography An Application Note UVP-AB-1000-02 #12;The GDS-8000 Gel on the overlayed scan. GEL ELECTROPHORESIS IMAGING, DOCUMENTATION AND ANALYSIS ... TODAY. #12;The introduction of the technique of electrophoresis in acrylamide or agarose gels was a major advance in nucleic acid technology

  17. 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.)

    PubMed Central

    Chatterjee, Moniya; Gupta, Sumanti; Bhar, Anirban; Das, Sampa

    2012-01-01

    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

  18. Laboratory and field validation of a Cry1Ab protein quantitation method for water.

    PubMed

    Strain, Katherine E; Whiting, Sara A; Lydy, Michael J

    2014-10-01

    The widespread planting of crops expressing insecticidal proteins derived from the soil bacterium Bacillus thuringiensis (Bt) has given rise to concerns regarding potential exposure to non-target species. These proteins are released from the plant throughout the growing season into soil and surface runoff and may enter adjacent waterways as runoff, erosion, aerial deposition of particulates, or plant debris. It is crucial to be able to accurately quantify Bt protein concentrations in the environment to aid in risk analyses and decision making. Enzyme-linked immunosorbent assay (ELISA) is commonly used for quantitation of Bt proteins in the environment; however, there are no published methods detailing and validating the extraction and quantitation of Bt proteins in water. The objective of the current study was to optimize the extraction of a Bt protein, Cry1Ab, from three water matrices and validate the ELISA method for specificity, precision, accuracy, stability, and sensitivity. Recovery of the Cry1Ab protein was matrix-dependent and ranged from 40 to 88% in the validated matrices, with an overall method detection limit of 2.1 ng/L. Precision among two plates and within a single plate was confirmed with a coefficient of variation less than 20%. The ELISA method was verified in field and laboratory samples, demonstrating the utility of the validated method. The implementation of a validated extraction and quantitation protocol adds consistency and reliability to field-collected data regarding transgenic products. PMID:25059137

  19. A quantitative recipe for engineering protein polymer nanoparticles

    PubMed Central

    Janib, S. Mohd; Pastuszka, M.; Aluri, S.; Folchman-Wagner, Z.; Hsueh, P-Y; Shi, P.; Yi-an; Cui, H.; MacKay, J.A.

    2013-01-01

    Protein polymers can assemble switchable nanostructures with emerging applications as biomaterials and nanomedicines. For example, above a critical micelle temperature (CMT) some elastin-like polypeptide (ELP) diblock copolymers assemble spherical nanoparticles, which may modulate cellular internalization and in vivo biodistribution. To achieve engineering-level control over their properties, this report explores a comprehensive library of ELP monoblock and diblock polymers. For the first time, we report that a surprisingly high core molecular weight is required for stable nanoparticle formation; furthermore, nanoparticle size depends on polymer molecular weight. A mathematical model was developed to characterize four ELP monoblock libraries and to predict the phase behavior of corresponding diblock copolymers. The CMT was almost entirely dependent on the hydrophobic core ELP, while the bulk phase transition temperature (Tt,bulk) depends predominantly on the hydrophilic block. Nanoparticle assembly was accompanied by a conversion in secondary structure of the hydrophobic block from random coil and beta-sheets to type-2 ? turns. For the first time, this report enables the rational design of ELP protein polymer nanoparticles with physico-chemico properties that will be suitable for biological applications. PMID:24511327

  20. A novel strategy for quantitative proteomics using isotope-coded protein labels.

    PubMed

    Schmidt, Alexander; Kellermann, Josef; Lottspeich, Friedrich

    2005-01-01

    Stable isotope labelling in combination with mass spectrometry has emerged as a powerful tool to identify and relatively quantify thousands of proteins within complex protein mixtures. Here we describe a novel method, termed isotope-coded protein label (ICPL), which is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on stable isotope tagging at the frequent free amino groups of isolated intact proteins, it is applicable to any protein sample, including extracts from tissues or body fluids, and compatible to all separation methods currently employed in proteome studies. The method showed highly accurate and reproducible quantification of proteins and yielded high sequence coverage, indispensable for the detection of post-translational modifications and protein isoforms. The efficiency (e.g. accuracy, dynamic range, sensitivity, speed) of the approach is demonstrated by comparative analysis of two differentially spiked proteomes. PMID:15602776

  1. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    PubMed Central

    Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

  2. Quantitative Glycomics Strategies*

    PubMed Central

    Mechref, Yehia; Hu, Yunli; Desantos-Garcia, Janie L.; Hussein, Ahmed; Tang, Haixu

    2013-01-01

    The correlations between protein glycosylation and many biological processes and diseases are increasing the demand for quantitative glycomics strategies enabling sensitive monitoring of changes in the abundance and structure of glycans. This is currently attained through multiple strategies employing several analytical techniques such as capillary electrophoresis, liquid chromatography, and mass spectrometry. The detection and quantification of glycans often involve labeling with ionic and/or hydrophobic reagents. This step is needed in order to enhance detection in spectroscopic and mass spectrometric measurements. Recently, labeling with stable isotopic reagents has also been presented as a very viable strategy enabling relative quantitation. The different strategies available for reliable and sensitive quantitative glycomics are herein described and discussed. PMID:23325767

  3. A statistical framework for protein quantitation in bottom-up MS-based proteomics

    SciTech Connect

    Karpievitch, Yuliya; Stanley, Jeffrey R.; Taverner, Thomas; Huang, Jianhua; Adkins, Joshua N.; Ansong, Charles; Heffron, Fred; Metz, Thomas O.; Qian, Weijun; Yoon, Hyunjin; Smith, Richard D.; Dabney, Alan R.

    2009-08-15

    ABSTRACT Motivation: Quantitative mass spectrometry-based proteomics requires protein-level estimates and confidence measures. Challenges include the presence of low-quality or incorrectly identified peptides and widespread, informative, missing data. Furthermore, models are required for rolling peptide-level information up to the protein level. Results: We present a statistical model for protein abundance in terms of peptide peak intensities, applicable to both label-based and label-free quantitation experiments. The model allows for both random and censoring missingness mechanisms and provides naturally for protein-level estimates and confidence measures. The model is also used to derive automated filtering and imputation routines. Three LC-MS datasets are used to illustrate the methods. Availability: The software has been made available in the open-source proteomics platform DAnTE (Polpitiya et al. (2008)) (http://omics.pnl.gov/software/). Contact: adabney@stat.tamu.edu

  4. Concerted, Rapid, Quantitative, and Site-Specific Dual Labeling of Proteins

    PubMed Central

    2014-01-01

    Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small molecules at physiological pH, temperature, and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-containing amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quantitative, site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, respectively. Protein double labeling in aqueous buffer at physiological pH, temperature, and pressure is quantitative in 30 min. PMID:24857040

  5. Label-free quantitative proteomics reveals differentially regulated proteins in experimental gingivitis.

    PubMed

    Bostanci, Nagihan; Ramberg, Per; Wahlander, Åsa; Grossman, Jonas; Jönsson, Daniel; Barnes, Virginia Monsul; Papapanou, Panos N

    2013-02-01

    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

  6. Quantitative changes in interleukin proteins following focal stroke in the rat

    Microsoft Academic Search

    Jeffrey J Legos; Robert G Whitmore; Joseph A Erhardt; Andrew A Parsons; Ronald F Tuma; Frank C Barone

    2000-01-01

    The aim of the present study was to quantitate the temporal changes in protein concentration for interleukin (IL)-1?, IL-1?, IL-1ra, and IL-6 from 1 h to 15 days following focal ischemia. Protein expression was evaluated by enzyme-linked immunosorbent assay utilizing newly available rat antibodies. There were no detectable basal levels of IL-1?, 1L-1?, or IL-6 in the sham-operated or non-ischemic

  7. Distinct and Overlapping Sets of SUMO1 and SUMO2 Target Proteins Revealed by Quantitative Proteomics

    Microsoft Academic Search

    Alfred C. O. Vertegaal; Jens S. Andersen; Stephen C. Ogg; Ronald T. Hay; Matthias Mann; Angus I. Lamond

    2006-01-01

    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

  8. Quantitation of tyrosine hydroxylase protein in the locus coeruleus from postmortem human brain

    Microsoft Academic Search

    Meng-Yang Zhu; Violetta Klimek; John W. Haycock; Gregory A. Ordway

    2000-01-01

    In this study, we developed an immuno-autoradiographic method to obtain quantitative estimates of tyrosine hydroxylase (TH) protein in tissue sections from post-mortem human brain. Protein from tissue sections containing the locus coeruleus (LC) was directly transferred to a polyvinylidene fluoride (PVDF) membrane. Immunoreactive TH on PVDF membranes was identified with optimized concentrations of TH antibody followed by application of [125I]labeled

  9. Quantitative Laser Diffraction Method for the Assessment of Protein Subvisible Particles

    PubMed Central

    Totoki, Shinichiro; Yamamoto, Gaku; Tsumoto, Kouhei; Uchiyama, Susumu; Fukui, Kiichi

    2015-01-01

    Laser diffraction (LD) has been recognized as a method for estimating particle size distribution. Here, a recently developed quantitative LD (qLD) system, which is an LD method with extensive deconvolution analysis, was employed for the quantitative assessment of protein particles sizes, especially aimed at the quantification of 0.2–10 ?m diameter subvisible particles (SVPs). The qLD accurately estimated concentration distributions for silica beads with diameters ranging from 0.2 to 10 ?m that have refractive indices similar to that of protein particles. The linearity of concentration for micrometer-diameter silica beads was confirmed in the presence of a fixed concentration of submicrometer diameter beads. Similarly, submicrometer-diameter silica beads could be quantified in the presence of micrometer-diameter beads. Subsequently, stir- and heat-stressed intravenous immunoglobulins were evaluated by using the qLD, in which the refractive index of protein particles that was determined experimentally was used in the deconvolution analysis. The results showed that the concentration distributions of protein particles in SVP size range differ for the two stresses. The number concentration of the protein particles estimated using the qLD agreed well with that obtained using flow microscopy. This work demonstrates that qLD can be used for quantitative estimation of protein aggregates in SVP size range. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:618–626, 2015 PMID:25449441

  10. Quantitative laser diffraction method for the assessment of protein subvisible particles.

    PubMed

    Totoki, Shinichiro; Yamamoto, Gaku; Tsumoto, Kouhei; Uchiyama, Susumu; Fukui, Kiichi

    2015-02-01

    Laser diffraction (LD) has been recognized as a method for estimating particle size distribution. Here, a recently developed quantitative LD (qLD) system, which is an LD method with extensive deconvolution analysis, was employed for the quantitative assessment of protein particles sizes, especially aimed at the quantification of 0.2-10 ?m diameter subvisible particles (SVPs). The qLD accurately estimated concentration distributions for silica beads with diameters ranging from 0.2 to 10 ?m that have refractive indices similar to that of protein particles. The linearity of concentration for micrometer-diameter silica beads was confirmed in the presence of a fixed concentration of submicrometer diameter beads. Similarly, submicrometer-diameter silica beads could be quantified in the presence of micrometer-diameter beads. Subsequently, stir- and heat-stressed intravenous immunoglobulins were evaluated by using the qLD, in which the refractive index of protein particles that was determined experimentally was used in the deconvolution analysis. The results showed that the concentration distributions of protein particles in SVP size range differ for the two stresses. The number concentration of the protein particles estimated using the qLD agreed well with that obtained using flow microscopy. This work demonstrates that qLD can be used for quantitative estimation of protein aggregates in SVP size range. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:618-626, 2015. PMID:25449441

  11. Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

    PubMed Central

    Barhoom, Sima; Kaur, Jaskiran; Cooperman, Barry S.; Smorodinsky, Nechama I.; Smilansky, Zeev; Ehrlich, Marcelo; Elroy-Stein, Orna

    2011-01-01

    We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection. PMID:21795382

  12. Using quantitative proteomics of Arabidopsis roots and leaves to predict metabolic activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins isolated from developing roots and leaves of Arabidopsis thaliana were separated by high-resolution two-dimensional (2-D) electrophoresis. The resulting 2-D proteome maps are markedly different. Quantitative analysis of root and leaf protein spot pairs revealed that in most instances ther...

  13. Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae

    PubMed Central

    Yin, Qing Yuan; de Groot, Piet W J; de Jong, Luitzen; Klis, Frans M; De Koster, Chris G

    2007-01-01

    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. PMID:17617218

  14. ITRAQ BASED PROTEIN QUANTITATIVE ANALYSIS OF THE CELL WALL PROTEOME OF PATHOGEN-INFECTED TOMATO LEAVES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The goal of this work is to use an iTRAQ-based shotgun approach to identify qualitative and quantitative changes in the extracellular proteome, or secretome, of tomato leaves following infection with the oomycete pathogen Phytophthora infestans. Proteins that are secreted by plants and microbes int...

  15. An immunoassay method for quantitative detection of proteins using single antibodies

    Microsoft Academic Search

    Shengliang Zhou; Xiaojuan Lu; Caifa Chen; Dongxu Sun

    2010-01-01

    A new immunoassay method called specific analyte labeling and recapture assay (SALRA) to quantitatively measure protein abundance was developed, and the assay conditions were optimized. The key features of this method include labeling the antigen bound to the capture antibody, eluting the labeled antigen, and recapturing it by the same capture antibody on the detection plate. The reporter molecules on

  16. High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography

    Microsoft Academic Search

    Mass Spectrometry; Hui Zhang; Eugene C. Yi; Xiao-jun Li; Parag Mallick; Karen S. Kelly-Spratt; Christophe D. Masselon; David G. Camp II; Richard D. Smith; Christopher J. Kemp; Ruedi Aebersold

    It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this informa- tion can be extracted via quantitative proteomic measure- ments. Suitable proteomic techniques need to be sensi- tive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins,

  17. Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS.

    PubMed

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

    2014-10-01

    Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by µbore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism. PMID:25059147

  18. Identification of proteins associated with Aha1 in HeLa cells by quantitative proteomics.

    PubMed

    Sun, Liang; Hartson, Steven D; Matts, Robert L

    2015-05-01

    The identification of the activator of heat shock protein 90 (Hsp90) ATPase's (Aha1) protein-protein interaction (PPI) network will provide critical insights into the relationship of Aha1 with multi-molecular complexes and shed light onto Aha1's interconnections with Hsp90-regulated biological functions. Flag-tagged Aha1 was over-expressed in HeLa cells and isolated by anti-Flag affinity pull downs, followed by trypsin digestion and identification co-adsorbing proteins by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). A probability-based identification of Aha1 PPIs was generated from the LC-MS/MS analysis by using a relative quantification strategy, spectral counting (SC). By comparing the SC-based protein levels between Aha1 pull-down samples and negative controls, 164 Aha1-interacting proteins were identified that were quantitatively enriched in the pull-down samples over the controls. The identified Aha1-interacting proteins are involved in a wide number of intracellular bioprocesses, including DNA maintenance, chromatin structure, RNA processing, translation, nucleocytoplasmic and vesicle transport, among others. The interactions of 33 of the identified proteins with Aha1 were further confirmed by Western blotting, demonstrating the reliability of our affinity-purification-coupled quantitative SC-MS strategy. Our proteomic data suggests that Aha1 may participate in diverse biological pathways to facilitate Hsp90 chaperone functions in response to stress. PMID:25614414

  19. Identification of DNA damage checkpoint-dependent protein interactions in Saccharomyces cerevisiae using quantitative mass spectrometry

    PubMed Central

    Bastos de Oliveira, Francisco M.; Smolka, Marcus B.

    2014-01-01

    Summary The DNA damage checkpoint (DDC) is an evolutionarily conserved signaling pathway that is crucial to maintain genomic integrity. In response to DNA damage, DDC kinases are rapidly activated and phosphorylate an elaborate network of substrates involved in multiple cellular processes. An important role of the DDC response is to assemble protein complexes. However, for most of the DDC substrates, how the DDC-dependent phosphorylation modulates their network of interactions remains to be established. Here, we present a protocol for the identification of DDC-dependent protein-protein interactions based on Stable Isotope Labeling of Amino acids in Cell culture (SILAC) followed by affinity-tagged protein purification and quantitative mass spectrometry analysis. Based on a model study using Saccharomyces cerevisiae, we provide a method that can be generally applied to study the role of kinases in mediating protein-protein interactions. PMID:24791994

  20. Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for quantitative parallel reaction monitoring of peptide abundance and single-shot proteomic analysis of a human cell line.

    PubMed

    Sun, Liangliang; Zhu, Guijie; Mou, Si; Zhao, Yimeng; Champion, Matthew M; Dovichi, Norman J

    2014-09-12

    We coupled capillary zone electrophoresis (CZE) with an ultrasensitive electrokinetically pumped nanospray ionization source for tandem mass spectrometry (MS/MS) analysis of complex proteomes. We first used the system for the parallel reaction monitoring (PRM) analysis of angiotensin II spiked in 0.45mg/mL of bovine serum albumin (BSA) digest. A calibration curve was generated between the loading amount of angiotensin II and intensity of angiotensin II fragment ions. CZE-PRM generated a linear calibration curve across over 4.5 orders of magnitude dynamic range corresponding to angiotensin II loading amount from 2amole to 150fmole. The relative standard deviations (RSDs) of migration time were <4% and the RSDs of fragment ion intensity were ?20% or less except 150fmole angiotensin II loading amount data (?36% RSD). We further applied the system for the first bottom up proteomic analysis of a human cell line using CZE-MS/MS. We generated 283 protein identifications from a 1h long, single-shot CZE MS/MS analysis of the MCF7 breast cancer cell line digest, corresponding to ?80ng loading amount. The MCF7 digest was fractionated using a C18 solid phase extraction column; single-shot analysis of a single fraction resulted in 468 protein identifications, which is by far the largest number of protein identifications reported for a mammalian proteomic sample using CZE. PMID:25082526

  1. A simple quantitative method to study protein-lipopolysaccharide interactions by using liquid crystals.

    PubMed

    Das, Dibyendu; Sidiq, Sumyra; Pal, Santanu Kumar

    2015-03-16

    The interaction of proteins with endotoxins has divergent effects on lipopolysaccharide (LPS)-induced responses, which serve as a basis for many clinical and therapeutic applications. It is, therefore, important to understand these interactions from both theoretical and practical points of view. This paper advances the design of liquid crystal (LC)-based stimuli-responsive soft materials for quantitative measurements of LPS-protein binding events through interfacial ordering transition. Micrometer-thick films of LCs undergo easily visualized ordering transitions in response to proteins at LPS-aqueous interfaces of the LCs. The optical response of the LC changes from dark to bright after aqueous solutions of hemoglobin (Hb), bovine serum albumin (BSA), and lysozyme proteins (LZM) are in contact with a LPS-laden aqueous-LC interface. The effects of interactions of different proteins with LPS are also observed to cause the response of the LC to vary significantly from one to another; this indicates that manipulation of the protein-LPS binding affinity can provide the basis for a general, facile method to tune the LPS-induced responses of the LCs to interfacial phenomena. By measuring the optical retardation of the 4'-pentyl-4-cyanobiphenyl (5CB) LC, the binding affinity of the proteins (Hb, BSA, and LZM) towards LPS that leads to different orientational behavior at the aqueous interfaces of the LCs can be determined. The interaction of proteins with the LPS-laden monolayer is highest for LPS-Hb, followed by LPS-BSA, and least for LPS-LZM; this is in correlation with their increasing order of binding constants (LPS-Hb>LPS-BSA>LPS-LZM). The results presented herein pave the way for quantitative and multiplexed measurements of LPS-protein binding events and reveal the potential of the LC system to be used as quantitative LC-based, stimuli-responsive soft materials. PMID:25572441

  2. Quantitative and Functional Characterization of the Hyper-Conserved Protein of Prochlorococcus and Marine Synechococcus

    PubMed Central

    Zorz, Jackie K.; Joy, Andrew P.; Barnett, David A.; Johnson, Milo S.; Zhaxybayeva, Olga; Cockshutt, Amanda M.

    2014-01-01

    A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly. PMID:25360678

  3. Determination of the sites of posttranslational modifications in the charge isomers of bovine myelin basic protein by capillary electrophoresis-mass spectroscopy.

    PubMed

    Zand, R; Li, M X; Jin, X; Lubman, D

    1998-02-24

    The posttranslational modifications in each of the 18.5 kDa bovine myelin basic protein charge isomers C-1 to C-6 have been determined by the use of capillary electrophoresis-mass spectroscopy. The pattern of modifications is viewed as being unique to each charge isomer and is thought to reflect a specific placement and function for each isomer in the myelin membrane. Several of the sites of posttranslational phosphorylation were found to differ from a number of the reported sites that were phosphorylated in vitro by various kinases. These differences suggest that an extremely cautious approach be taken in identifying in vivo posttranslationally modified amino acid residues from residues that have been modified in vitro by various kinases. We have identified the following posttranslationally phosphorylated and deamidated, modified sites in the bovine MBP components C1-C6. C1 has no modification; C2 represents a deamidation of Gln 146; in C3, Thr 97 and Ser 164 are phosphorylated; in C4, Ser 54, Thr 97, and Ser 160 are phosphorylated; in C5 Ser 7, Ser 54, Thr 97, and Ser 164 are phosphorylated; and in C6, Ser 7, Ser 54, Thr 97, Ser 160, and Ser 164 are phosphorylated. PMID:9485392

  4. Electrophoresis in space at zero gravity

    NASA Technical Reports Server (NTRS)

    Bier, M.; Snyder, R. S.

    1974-01-01

    Early planning for manufacturing operations in space include the use of electrophoresis for purification and separation of biological materials. Greatly simplified electrophoresis apparatus have been flown in the Apollo 14 and 16 missions to test the possibility of stable liquid systems in orbit. Additionally, isoelectric focusing and isotachophoresis are of particular interest as they offer very high resolution and have self-sharpening boundaries. The value of possible space electrophoresis is substantial. For example, present technology permits large fractionation of only a few of blood proteins many fractions, and separated cell populations are needed for research.

  5. A sensitive and specific ELISA immunocapture assay for rapid quantitation of influenza A\\/H3N2 neuraminidase protein

    Microsoft Academic Search

    L Gerentes; N Kessler; M Aymard

    1998-01-01

    Both HA and NA proteins elicit antibodies which have been shown to be capable of altering the course of infection. Nevertheless, while influenza virus vaccine standardization involves hemagglutinin (HA) and neuraminidase (NA) in terms of antigenic characterization, only HA protein quantitation is undertaken. An immunocapture ELISA (EIA) is described for N2 NA quantitation, based on the use of a highly

  6. A novel diazoresin/poly(N-vinyl aminobutyric acid) covalent capillary coating for the analysis of proteins by capillary electrophoresis.

    PubMed

    Yu, Bing; Jiao, Mingming; Cong, Hailin; Shu, Xi; Yang, Shujing

    2014-03-01

    A novel method for the preparation of covalently linked capillary coatings of poly(N-vinyl aminobutyric acid) (PVAA) obtained from hydrolyzed polyvinylpyrrolidone was demonstrated using photosensitive diazoresin (DR) as a coupling agent. A layer-by-layer self-assembled film of DR and PVAA based on ionic bonding was first fabricated on the inner wall of capillary, then ionic bonding was converted into covalent bonding after treatment with UV light through a unique photochemical reaction of DR. The covalently bonded coatings suppressed protein adsorption on the inner surface of the capillary, and thus a baseline separation of lysozyme, cytochrome c, BSA, amyloglucosidase, and myoglobin was achieved using CE. Compared with bare capillary or noncovalently bonded DR/PVAA coatings, the covalently linked DR/PVAA capillary coatings not only improved the CE separation performance for proteins, but also exhibited good stability and repeatability. Due to the replacement of the highly toxic and moisture-sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide a green and easy way to make covalently coated capillaries for CE. PMID:24449602

  7. Tissue-specific ribosomal protein composition.

    PubMed

    Hoffman, W L; Dowben, R M

    1979-12-31

    Membrane-bound and free polysomes from murine liver and kidney were isolated under identical conditions and their ribosomal proteins were compared by two-dimensional gel electrophoresis. The results demonstrate that these ribosome subpopulations are quantitatively and qualitatively similar except for the presence of one additional protein in the kidney-bound polysomal fraction. PMID:530271

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

    E-print Network

    Vagenende, Vincent

    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 ...

  9. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways.

    PubMed

    Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

    2014-07-17

    Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct ?-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement. PMID:25036637

  10. Quantitative studies of the effect of HTLV-I Tax protein on CREB protein--DNA binding.

    PubMed Central

    Anderson, M G; Dynan, W S

    1994-01-01

    The human T-cell leukemia virus type I (HTLV-I) Tax protein increases the DNA binding activity of a number of different host cell transcription factors, including the cyclic AMP response element binding protein (CREB). We have performed quantitative studies of CREB binding in the presence and absence of Tax in an attempt to gain insight into the mechanism of the Tax effect. Enhancement of binding occurred over a wide range of CREB concentrations, but sharply increased at the lowest concentrations tested. The data are best explained by a two-step binding model where Tax changes the apparent equilibrium constants for both a CREB-CREB dimerization step and a (CREB)2-DNA binding step. We used the model to perform a quantitative analysis of the binding of CREB to DNA that had been mutated at positions flanking the core CREB recognition site. Results suggest that there are altered or more extensive DNA-protein contacts at these positions in the presence of Tax. We also used the model to analyze differences in the interaction of Tax with nonphosphorylated and protein kinase A-phosphorylated CREB protein. There was no significant change in the behavior of CREB upon phosphorylation. Images PMID:8065935

  11. Kidney Cell Electrophoresis

    NASA Technical Reports Server (NTRS)

    Todd, P.

    1985-01-01

    Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated, ground support in the form of analytical cell electrophoresis and flow cytometry was provided and cells returned from space flight were analyzed. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. The protocol established and utilized is given.

  12. Identification of plant mitochondrial proteins: A procedure linking two-dimensional gel electrophoresis to protein sequencing from PVDF membranes using a FastBlot cycle

    Microsoft Academic Search

    Bryan Dunbar; Thomas E. Elthon; John C. Osterman; Beth A. Whitaker; S. Brian Wilson

    1997-01-01

    Identification of the 329 spots visible in 2D gels of plant mitochondrial proteins is a challenge. This paper describes a\\u000a 2D mini-gel protocol involving free-radical scavengers and purified reagents to make it compatible with protein sequencing,\\u000a and evaluates its performance. The paper also describes a “FastBlot” sequencing cycle with the cycle time for protein sequencing\\u000a from PVDF membranes reduced to

  13. Methods for Peptide and Protein Quantitation by Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry*

    PubMed Central

    Zhang, Haixia; Liu, Qinfeng; Zimmerman, Lisa J.; Ham, Amy-Joan L.; Slebos, Robbert J. C.; Rahman, Jamshedur; Kikuchi, Takefume; Massion, Pierre P.; Carbone, David P.; Billheimer, Dean; Liebler, Daniel C.

    2011-01-01

    Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and ?-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633–641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20–30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications. PMID:21357624

  14. Nine surface plasmon resonance assays for specific protein quantitation during cell culture and process development.

    PubMed

    Frostell, Sa; Mattsson, Anna; Eriksson, Sa; Wallby, Elisabeth; Kärnhall, Johan; Illarionova, Nina B; Estmer Nilsson, Camilla

    2015-05-15

    Quantitation of protein is essential during pharmaceutical development, and a variety of methods and technologies for determination of total and specific protein concentration are available. Here we describe the development of a streamlined assay platform for specific quantitation assays using surface plasmon resonance (SPR) technology. A total of nine different assays were developed using similar conditions, of which eight assays were for quantitation of different human blood plasma proteins (IgG, IgG1-4 subclasses, IgA, transferrin, and albumin) from a chromatography-based IgG plasma process. Lastly, an assay for monitoring the concentration of a recombinant monoclonal antibody during 13days of CHO cell culturing was developed. Assay performances were compared with enzyme-linked immunosorbent assay (ELISA), nephelometry, ARCHITECT, and Cobas c501. SPR assays were shown to have higher sensitivity than analysis using nephelometry, ARCHITECT, and Cobas and to have significantly lower analysis and hands-on time compared with ELISA. Furthermore, the SPR assays were robust enough to be used for up to 12days, allowing specific protein concentration measurement of a sample to be completed at line within 10min. Using the same platform with only few varied parameters between different assays has saved time in the lab as well as for evaluation and presentation of results. PMID:25700863

  15. A Miniaturized Technique for Assessing Protein Thermodynamics and Function Using Fast Determination of Quantitative Cysteine Reactivity

    PubMed Central

    Isom, Daniel G.; Marguet, Philippe R.; Oas, Terrence G.; Hellinga, Homme W.

    2010-01-01

    Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity (fQCR), which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics. PMID:21387407

  16. Advantages of quantitative affinity chromatography for the analysis of solute interaction with membrane proteins.

    PubMed

    Lundqvist, A; Lundahl, P

    2001-10-30

    The use of membrane proteins as chromatographic stationary phases for the quantitation of biospecific interaction between the proteins and solutes is reviewed. This method is one among the few where a membrane protein is immobilized for repeated analyses of solute binding. To our knowledge, five transmembrane proteins have been immobilized in chromatographic matrices: the glucose and nucleoside transporters from human red blood cells, the photosynthetic reaction center from Rhodobacter sphaeroides, the nicotinic acetylcholine receptor from rat brain and a recombinant P-glycoprotein. Proteoliposomes and membrane vesicles have thereby been entrapped in size-exclusion beads, such as Superdex 200, and membrane proteins have been adsorbed on 'immobilized artificial membrane' monolayers of lipid analogs grafted to silica beads. Encouragingly, immobilized glucose transporter and P-glycoprotein showed constant interactant affinities for months. Analysis is done in the frontal mode at equilibrium because there is no separation between bound and free ligand. Both the affinity constant, which generally coincides with the corresponding constant determined by use of nonchromatographic methods, and the amount of active binding sites are obtained. The method has been successfully applied to functional analysis of membrane proteins in cells or reconstituted in lipid mono- or bilayers, screening of low-molecular interactants, investigation of protein-protein interaction and studies of effects of physico-chemical parameters on solute-protein interaction. The analyses require sensitive detection of the analyte and matching between amount of binding sites and affinity. PMID:11694298

  17. Seasonal liver protein differences in a hibernator revealed by quantitative proteomics using whole animal isotopic labeling

    PubMed Central

    Rose, J. Cameron; Epperson, L. Elaine; Carey, Hannah V.; Martin, Sandra L.

    2011-01-01

    Hibernation is an energy-saving strategy used by diverse species of mammals to survive winter. It is characterized by cycles between multi-day periods of torpor with low body temperature (Tb), and short periods of rapid, spontaneous rewarming. The ability to retain cellular integrity and function throughout torpor and rewarming is a key attribute of hibernation. Livers from winter hibernators are resistant to cellular damage induced by cold storage followed by warm reperfusion. Identifying proteins that differ between the summer-sensitive and winter-protected phenotypic states is one useful approach that may elucidate the molecular mechanisms that underlie this protection. Here we employ a novel quantitative proteomics screening strategy whereby a newly-weaned 13-lined ground squirrel was metabolically labeled by ingesting heavy-isotope substituted (15N) Spirulina. The liver protein extract from this animal provided a common reference for quantitative evaluation of protein differences by its addition to extracts from pooled samples of summer active (SA) or winter entrance (Ent) phase hibernating ground squirrels. We identified 61 significantly different proteins between the two groups and compared them to proteins identified previously in the same samples using 2D gels. Of the 20 proteins common to the two datasets, the direction and magnitude of their differences were perfectly concordant for 18, providing confidence that both sets of altered proteins reflect bona fide differences between the two physiological states. Furthermore, the 41 novel proteins recovered in this study included many new enzymes in pathways identified previously: specifically, additional enzymes belonging to the urea cycle, amino acid and carbohydrate degradation, and lipid biosynthetic pathways were decreased, whereas enzymes involved in ketone body synthesis, fatty acid utilization, protein synthesis and gluconeogenesis were increased in the samples from entrance hibernators compared to summer active animals, providing additional specific evidence for the importance of these pathways in the hibernating phenotype. PMID:21481655

  18. Protein Quantitative Trait Loci Identify Novel Candidates Modulating Cellular Response to Chemotherapy

    PubMed Central

    Gorsic, Lidija K.; Antao, Nirav N.; Wong, Shan S.; Chung, Sophie H.; Gill, Daniel F.; Im, Hae K.; Myers, Jamie L.; White, Kevin P.; Jones, Richard Baker; Dolan, M. Eileen

    2014-01-01

    Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p?0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms. PMID:24699359

  19. Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics*

    PubMed Central

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

    2013-01-01

    Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423

  20. Quantitative study of protein-protein interactions in live cell by dual-color fluorescence correlation spectroscopy.

    PubMed

    Padilla-Parra, Sergi; Audugé, Nicolas; Coppey-Moisan, Maïté; Tramier, Marc

    2014-01-01

    Dual-color FCS is a powerful method to monitor protein-protein interactions in living cells. The main idea is based on the cross-correlation analysis of temporal fluorescence intensity fluctuations of two fluorescent proteins to obtain their co-diffusion and relative concentration. But, when performing these experiments, the spectral overlap in the emission of the two colors produces an artifact that corrupts the cross-correlation data: spectral bleed-through. We have shown that problems with cross talk are overcome with Fluorescence Lifetime Correlation Spectroscopy (FLCS). FLCS applied to dual-color cross-correlation, utilizing for example eGFP and mCherry fluorescent proteins, allows the determination of protein-protein interactions in living cells without the need of spectral bleed-through calibration. Here, we present in detail how this methodology can be implemented using a commercial setup (Microtime from PicoQuant, SP8 SMD from Leica or any conventional confocal with PicoQuant TCSPC module, and also with a Becker and Hickl TCSPC module). The dual-color FLCS experimental procedure where the different laser intensities do not have to be controlled during the experiment constitutes a very powerful technique to quantitatively study protein interactions in live samples. PMID:24108650

  1. Quantitative time-resolved measurement of membrane protein-ligand interactions using microcantilever array sensors

    NASA Astrophysics Data System (ADS)

    Braun, Thomas; Ghatkesar, Murali Krishna; Backmann, Natalija; Grange, Wilfried; Boulanger, Pascale; Letellier, Lucienne; Lang, Hans-Peter; Bietsch, Alex; Gerber, Christoph; Hegner, Martin

    2009-03-01

    Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor-the FhuA receptor of Escherichia coli-is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein-ligand interactions in a micro-array format.

  2. Quantitative variability of 342 plasma proteins in a human twin population.

    PubMed

    Liu, Yansheng; Buil, Alfonso; Collins, Ben C; Gillet, Ludovic C J; Blum, Lorenz C; Cheng, Lin-Yang; Vitek, Olga; Mouritsen, Jeppe; Lachance, Genevieve; Spector, Tim D; Dermitzakis, Emmanouil T; Aebersold, Ruedi

    2015-01-01

    The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2-7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis-SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood-based biomarker studies. PMID:25652787

  3. Quantitative variability of 342 plasma proteins in a human twin population

    PubMed Central

    Liu, Yansheng; Buil, Alfonso; Collins, Ben C; Gillet, Ludovic CJ; Blum, Lorenz C; Cheng, Lin-Yang; Vitek, Olga; Mouritsen, Jeppe; Lachance, Genevieve; Spector, Tim D; Dermitzakis, Emmanouil T; Aebersold, Ruedi

    2015-01-01

    The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis-SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood-based biomarker studies. PMID:25652787

  4. A Quantitative Proteomics-based Competition Binding Assay to Characterize pITAM-Protein Interactions

    PubMed Central

    Hu, Lianghai; Yang, Li; Lipchik, Andrew M.; Geahlen, Robert L.; Parker, Laurie L.; Tao, W. Andy

    2013-01-01

    Characterization of ligand-protein binding is of crucial importance in drug discovery. Classical competition binding assays measure the binding of a labeled ligand in the presence of various concentrations of unlabeled ligand, and typically use single purified proteins. Here we introduce a high-throughput approach to study ligand-protein interactions by coupling competition binding assays with mass spectrometry-based quantitative proteomics. Using a phosphorylated immunoreceptor tyrosine-based activation motif (pITAM) peptide as a model, we characterized pITAM-interacting partners in human lymphocytes. The shapes of competition binding curves of various interacting partners constructed in a single set of quantitative proteomics experiments reflect relative affinities for the pITAM peptide. This strategy can provide an efficient approach to distinguish specific interacting partners, including two signaling kinases possessing tandem SH2 domains, SYK and ZAP-70, as well as other SH2 domain containing proteins such as CSK and PI3K, from contaminants and to measure relative binding affinities of multiple proteins in a single experiment. PMID:23611696

  5. Quantitative Phosphoproteomics of Cytotoxic T Cells to Reveal Protein Kinase D 2 Regulated Networks*

    PubMed Central

    Navarro, María N.; Goebel, Juergen; Hukelmann, Jens L.; Cantrell, Doreen A.

    2014-01-01

    The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labeling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T-cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription, and translation. In other cell types, PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages. PMID:25266776

  6. Qualitative and Quantitative Detection of Protein and Genetic Traits in Genetically Modified Food

    Microsoft Academic Search

    P. Markoulatos; N. Siafakas; A. Papathoma; E. Nerantzis; B. Betzios; V. Dourtoglou; M. Moncany

    2004-01-01

    Due to the market introduction of genetically modified organisms (GMOs) in crops, foods, and ingredients, legislation worldwide came face to face with the question of the use and labeling requirements on GMO crops and their derivatives. In this review, protein- and DNA-based methods, such as enzyme-linked immunosorbent assay, western blots, and qualitative and quantitative polymerase chain reaction PCR (Q-PCR) are

  7. Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins

    Microsoft Academic Search

    ESA STENBERG; HAKAN ROOS; CSABA URBANICZKY

    1991-01-01

    A methodology to correlate the absolute surface concentration of protein to the surface plasmon resonance (SPR) response is described. The thickness and the optical constants for each layer on the sensor chip used were determined with different optical techniques. In a flow injection system, the steady- state SPR response was correlated to the absolute amount of radiolabeled protein adsorbed by

  8. Automatic multiple applicator electrophoresis

    NASA Technical Reports Server (NTRS)

    Grunbaum, B. W.

    1977-01-01

    Easy-to-use, economical device permits electrophoresis on all known supporting media. System includes automatic multiple-sample applicator, sample holder, and electrophoresis apparatus. System has potential applicability to fields of taxonomy, immunology, and genetics. Apparatus is also used for electrofocusing.

  9. Electrophoresis of biological materials

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The selection of biological products was studied for electrophoresis in space. Free flow electrophoresis, isoelectric focusing, and isotachophoresis are described. The candidates discussed include: immunoglobulins and gamma globulins; isolated islet of langerhans from pancreas; bone marrow; tumor cells; kidney cells, cryoprecipitate; and column separated cultures.

  10. Integrated protocol for reliable and fast quantification and documentation of electrophoresis gels.

    PubMed

    Rehbein, Peter; Schwalbe, Harald

    2015-06-01

    Quantitative analysis of electrophoresis gels is an important part in molecular cloning, as well as in protein expression and purification. Parallel quantifications in yield and purity can be most conveniently obtained from densitometric analysis. This communication reports a comprehensive, reliable and simple protocol for gel quantification and documentation, applicable for single samples and with special features for protein expression screens. As major component of the protocol, the fully annotated code of a proprietary open source computer program for semi-automatic densitometric quantification of digitized electrophoresis gels is disclosed. The program ("GelQuant") is implemented for the C-based macro-language of the widespread integrated development environment of IGOR Pro. PMID:25514201

  11. Quantitative detection of zeta-chain-associated protein 70 expression in chronic lymphocytic leukemia

    PubMed Central

    Zhu, Peixuan; Degheidy, Heba A.; Marti, Gerald E.; Li, Shuhong; Abbasi, Fatima; Wiestner, Adrian; Amstutz, Platte; Tang, Cha-Mei

    2013-01-01

    Overexpression of zeta-chain-associated protein 70 (ZAP-70) was recently recognized as an independent prognostic marker for the aggressive form of chronic lymphocytic leukemia (CLL). The objective of this study was to demonstrate the feasibility and implementation of quantitative detection of ZAP-70 protein in B cells to clearly distinguish patients with CLL with the aggressive form of the disease. B cells were isolated from patient blood and lysed. Released ZAP-70 protein was detected using an immunomagnetic fluorescence assay. The assay protocol was developed using Jurkat cells and recombinant ZAP-70 (rZAP-70). The limit of detection was determined to be lower than 125 Jurkat cells and 39 pg of rZAP-70 protein. The signal response was linear over a wide dynamic range, from 125 to 40 000 Jurkat cells per test (R2 = 0.9987) and from 0 to 40 000 pg rZAP-70 protein per test (R2 = 0.9928). The results from 20 patients with CLL correlated strongly with flow cytometry analysis. Concordance between the two methods for positive and negative results was 100% (7/7) and 92% (12/13), respectively, while the overall concordance between the two methods was 95%. The assay reported here is a simple, reliable and reproducible method for quantitative detection of ZAP-70 in patient leukemic cells, without the need for cell fixation or permeabilization. The ZAP-70 signal was linear over a wide dynamic range, which we believe enables quantitative assessment of small changes in ZAP-70 expression over the course of the disease and in response to therapeutic intervention. PMID:22839722

  12. Improved Protein Arrays for Quantitative Systems Analysis of the Dynamics of Signaling Pathway Interactions

    SciTech Connect

    YANG, CHIN-RANG [NHLBI, NIH] [NHLBI, NIH

    2013-12-11

    Astronauts and workers in nuclear plants who repeatedly exposed to low doses of ionizing radiation (IR, <10 cGy) are likely to incur specific changes in signal transduction and gene expression in various tissues of their body. Remarkable advances in high throughput genomics and proteomics technologies enable researchers to broaden their focus from examining single gene/protein kinetics to better understanding global gene/protein expression profiling and biological pathway analyses, namely Systems Biology. An ultimate goal of systems biology is to develop dynamic mathematical models of interacting biological systems capable of simulating living systems in a computer. This Glue Grant is to complement Dr. Boothman’s existing DOE grant (No. DE-FG02-06ER64186) entitled “The IGF1/IGF-1R-MAPK-Secretory Clusterin (sCLU) Pathway: Mediator of a Low Dose IR-Inducible Bystander Effect” to develop sensitive and quantitative proteomic technology that suitable for low dose radiobiology researches. An improved version of quantitative protein array platform utilizing linear Quantum dot signaling for systematically measuring protein levels and phosphorylation states for systems biology modeling is presented. The signals are amplified by a confocal laser Quantum dot scanner resulting in ~1000-fold more sensitivity than traditional Western blots and show the good linearity that is impossible for the signals of HRP-amplification. Therefore this improved protein array technology is suitable to detect weak responses of low dose radiation. Software is developed to facilitate the quantitative readout of signaling network activities. Kinetics of EGFRvIII mutant signaling was analyzed to quantify cross-talks between EGFR and other signaling pathways.

  13. Polyacrylamide gel electrophoretic methods in the separation of structural muscle proteins.

    SciTech Connect

    Barany, K.; Barany, M.; Giometti, C. S.; Center for Mechanistic Biology and Biotechnology; Univ. of Illinois at Chicago

    1995-04-28

    Polyacrylamide gel electrophoresis plays a major role in analyzing the function of muscle structural proteins. This review describes one- and two-dimensional gel electrophoretic methods for qualitative and quantitative investigation of the muscle proteins, with special emphasis on determination of protein phosphorylation. The electrophoretic studies established the subunit structures of the muscle proteins, characterized their multiple forms, revealed changes in subunit composition or shifts in isoform distribution of specific proteins during development, upon stimulation or denervation of the muscle. Protein phosphorylation during muscle contraction is preferentially studied by two-dimensional gel electrophoresis. The same method demonstrated protein alterations in human neuromuscular diseases.

  14. Genome-scale quantitative characterization of bacterial protein localization dynamics throughout the cell cycle.

    PubMed

    Kuwada, Nathan J; Traxler, Beth; Wiggins, Paul A

    2015-01-01

    Bacterial cells display both spatial and temporal organization, and this complex structure is known to play a central role in cellular function. Although nearly one-fifth of all proteins in Escherichia coli localize to specific subcellular locations, fundamental questions remain about how cellular-scale structure is encoded at the level of molecular-scale interactions. One significant limitation to our understanding is that the localization behavior of only a small subset of proteins has been characterized in detail. As an essential step toward a global model of protein localization in bacteria, we capture and quantitatively analyze spatial and temporal protein localization patterns throughout the cell cycle for nearly every protein in E. coli that exhibits nondiffuse localization. This genome-scale analysis reveals significant complexity in patterning, notably in the behavior of DNA-binding proteins. Complete cell-cycle imaging also facilitates analysis of protein partitioning to daughter cells at division, revealing a broad and robust assortment of asymmetric partitioning behaviors. PMID:25353361

  15. Genome-scale quantitative characterization of bacterial protein localization dynamics throughout the cell cycle

    PubMed Central

    Kuwada, Nathan J; Traxler, Beth; Wiggins, Paul A

    2015-01-01

    Bacterial cells display both spatial and temporal organization, and this complex structure is known to play a central role in cellular function. Although nearly one-fifth of all proteins in Escherichia coli localize to specific subcellular locations, fundamental questions remain about how cellular-scale structure is encoded at the level of molecular-scale interactions. One significant limitation to our understanding is that the localization behavior of only a small subset of proteins has been characterized in detail. As an essential step toward a global model of protein localization in bacteria, we capture and quantitatively analyze spatial and temporal protein localization patterns throughout the cell cycle for nearly every protein in E. coli that exhibits nondiffuse localization. This genome-scale analysis reveals significant complexity in patterning, notably in the behavior of DNA-binding proteins. Complete cell-cycle imaging also facilitates analysis of protein partitioning to daughter cells at division, revealing a broad and robust assortment of asymmetric partitioning behaviors. PMID:25353361

  16. A Statistical Framework for Protein Quantitation in Bottom-Up MS-Based Proteomics

    SciTech Connect

    Karpievitch, Yuliya; Stanley, Jeffrey R.; Taverner, Thomas; Huang, Jianhua; Adkins, Joshua N.; Ansong, Charles; Heffron, Fred; Metz, Thomas O.; Qian, Weijun; Yoon, Hyunjin; Smith, Richard D.; Dabney, Alan R.

    2009-08-15

    Motivation: Quantitative mass spectrometry-based proteomics requires protein-level estimates and associated confidence measures. Challenges include the presence of low quality or incorrectly identified peptides and informative missingness. Furthermore, models are required for rolling peptide-level information up to the protein level. Results: We present a statistical model that carefully accounts for informative missingness in peak intensities and allows unbiased, model-based, protein-level estimation and inference. The model is applicable to both label-based and label-free quantitation experiments. We also provide automated, model-based, algorithms for filtering of proteins and peptides as well as imputation of missing values. Two LC/MS datasets are used to illustrate the methods. In simulation studies, our methods are shown to achieve substantially more discoveries than standard alternatives. Availability: The software has been made available in the opensource proteomics platform DAnTE (http://omics.pnl.gov/software/). Contact: adabney@stat.tamu.edu Supplementary information: Supplementary data are available at Bioinformatics online.

  17. Quantitative Reduction of the TCR Adapter Protein SLP-76 Unbalances Immunity and Immune Regulation.

    PubMed

    Siggs, Owen M; Miosge, Lisa A; Daley, Stephen R; Asquith, Kelly; Foster, Paul S; Liston, Adrian; Goodnow, Christopher C

    2015-03-15

    Gene variants that disrupt TCR signaling can cause severe immune deficiency, yet less disruptive variants are sometimes associated with immune pathology. Null mutations of the gene encoding the scaffold protein Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76), for example, cause an arrest of T cell positive selection, whereas a synthetic membrane-targeted allele allows limited positive selection but is associated with proinflammatory cytokine production and autoantibodies. Whether these and other enigmatic outcomes are due to a biochemical uncoupling of tolerogenic signaling, or simply a quantitative reduction of protein activity, remains to be determined. In this study we describe a splice variant of Lcp2 that reduced the amount of wild-type SLP-76 protein by ?90%, disrupting immunogenic and tolerogenic pathways to different degrees. Mutant mice produced excessive amounts of proinflammatory cytokines, autoantibodies, and IgE, revealing that simple quantitative reductions of SLP-76 were sufficient to trigger immune dysregulation. This allele reveals a dose-sensitive threshold for SLP-76 in the balance of immunity and immune dysregulation, a common disturbance of atypical clinical immune deficiencies. PMID:25662996

  18. High Throughput Quantitative Analysis of Serum Proteins using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry

    SciTech Connect

    Zhang, Hui; Yi, Eugene C.; Li, Xiao-jun; Mallick, Parag; Kelly-Spratt, Karen S.; Masselon, Christophe D.; Camp, David G.; Smith, Richard D.; Kemp, Christopher; Aebersold, Ruedi

    2005-02-01

    It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease, and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible and robust to detect potential biomarkers below the level of highly expressed proteins, to generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. In this paper, we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these, no de-glycosylated peptides by LC-ESI-MS, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared to their control littermates.

  19. Quantitative analysis of cohesin complex stoichiometry and SMC3 modification-dependent protein interactions

    PubMed Central

    Ding, Chen; Li, Yehua; Kim, Beom-Jun; Malovannaya, Anna; Jung, Sung Yun; Wang, Yi; Qin, Jun

    2011-01-01

    Cohesin is a protein complex that plays an essential role in pairing replicated sister chromatids during cell division 1-3. The vertebrate cohesin complex consists of four core components including structure maintenance of chromosomes proteins SMC1 and SMC3, RAD21 and SA2/SA1. Extensive research suggests that cohesin traps the sister chromatids by a V-shaped SMC1/SMC3 heterodimer bound to the RAD21 protein 4 that closes the ring. Accordingly, the single ‘ring’ model proposes that two sister chromatids are trapped in a single ring that is composed of one molecule each of the 4 subunits. However, evidence also exists for alternative models. The hand-cuff model suggests that each sister chromatid is trapped individually by two rings that are joined through the shared SA1/SA2 subunit. We report here the determination of cohesin subunit stoichiometry of endogenous cohesin complex by quantitative mass spectrometry. Using qConCAT-based isotope labeling, we show that the cohesin core complex contains equimolar of the 4 core components, suggesting that each cohesin ring is closed by one SA1/SA2 molecule. Furthermore, we applied this strategy to quantify post-translational modification-dependent cohesin interactions. We demonstrate that quantitative mass spectrometry is a powerful tool for measuring stoichiometry of endogenous protein core complex. PMID:21699228

  20. Quantitative analysis of the protein corona on FePt nanoparticles formed by transferrin binding

    PubMed Central

    Jiang, Xiue; Weise, Stefan; Hafner, Margit; Röcker, Carlheinz; Zhang, Feng; Parak, Wolfgang J.; Nienhaus, G. Ulrich

    2010-01-01

    Nanoparticles are finding a rapidly expanding range of applications in research and technology, finally entering our daily life in medical, cosmetic or food products. Their ability to invade all regions of an organism including cells and cellular organelles offers new strategies for medical diagnosis and therapy (nanomedicine), but their safe use requires a deep knowledge about their interactions with biological systems at the molecular level. Upon incorporation, nanoparticles are exposed to biological fluids from which they adsorb proteins and other biomolecules to form a ‘protein corona’. These nanoparticle–protein interactions are still poorly understood and quantitative studies to characterize them remain scarce. Here we have quantitatively analysed the adsorption of human transferrin onto small (radius approx. 5 nm) polymer-coated FePt nanoparticles by using fluorescence correlation spectroscopy. Transferrin binds to the negatively charged nanoparticles with an affinity of approximately 26 µM in a cooperative fashion and forms a monolayer with a thickness of 7 nm. By using confocal fluorescence microscopy, we have observed that the uptake of FePt nanoparticles by HeLa cells is suppressed by the protein corona compared with the bare nanoparticles. PMID:19776149

  1. An Investigation on Gel Electrophoresis with Quantum Dots End-labeled DNA

    E-print Network

    Chen, Xiaojia

    2009-05-15

    Invented in the 1950s, gel electrophoresis has now become a routine analytical method to verify the size of nucleic acids and proteins in molecular biology labs. Conventional gel electrophoresis can successfully separate DNA fragments from several...

  2. Abstract--This work addresses the segmentation of two-dimensional polyacrylamide gel electrophoresis images

    E-print Network

    Athens, University of

    -PAGE Images, Overlapping Spots I. INTRODUCTION wo-dimensional polyacrylamide gel electrophoresis (2- D PAGEAbstract--This work addresses the segmentation of two- dimensional polyacrylamide gel electrophoresis images containing overlapping protein spots. A novel segmentation approach is proposed, which

  3. Proteomic analysis of rice after different seed space flights by two-dimensional difference electrophoresis

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Liang, Shujian; Sun, Yeqing

    To investigate the biological effects of space environment in rice plants, proteomic profiles of six rice cultivars growing after twice different seed space flights were analyzed by two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS). Over 1500 protein spots were detected in each paired space/ground-control comparison and more than 800 protein spots were reproducible across all the samples. Six proteins including peroxiredoxin and rubisco were found significantly changed in most of the six cultivars after both of the seed space flights, indicating they might be associated with the responses of rice cells to the space environment. Cluster analyses were also applied using the quantitative protein expression data: cultivar hierarchical clustering and principal component analysis both indicated that the rice proteome changed its expression profiles after seed space environment exposures while protein hierarchical clustering revealed that there might be a decrease of protein expression in rice plants after seed space flights.

  4. Preparative electrophoresis experiment design

    NASA Technical Reports Server (NTRS)

    Thiehler, A.

    1972-01-01

    A multifaceted study supporting the NASA programs to develop a space electrophoresis capability has been conducted. The study involved principally the technique of continuous free electrophoresis. It comprised a critical review of the art, study of new techniques for enhancing resolution and stability, and construction and initial testing of a high resolution cell. The effort resulted in a significant advance in free electrophoresis technique. It has provided also a much improved base for developments exploiting the added advantages of a zero-gravity environment.

  5. Quantitative changes in sets of proteins as markers of biological response

    SciTech Connect

    Giometti, C.S.; Taylor, J.; Gemmell, M.A.; Tollaksen, S.L. (Argonne National Lab., IL (USA)); Lalwani, N.D.; Reddy, J.K. (Northwestern Univ., Chicago, IL (USA))

    1990-01-01

    Exposure to either physical or chemical insults triggers a cascade of bio-chemical events within the target cell. This response requires adjustment within the protein population of the cell, some proteins becoming more abundant (those involved in the cellular response), others less abundant (those not required or counterproductive to the response). Thus, quantitative changes in the global protein population of an exposed biological system may well serve as an indicator of exposure, provided the alterations observed are selective and dose-dependent. In this paper we present results from a study in which liver protein changes induced by exposure of mice to chemicals known to cause peroxisome proliferation and subsequent hepatocellular carcinoma where monitored. Clofibrate, and its chemical analog ciprofibrate, are hypolipidemic drugs. Di-(ethylhexyl)phthalate (DEHP) is a plasticizer used widely in disposable containers for blood products. WY-14643 is a chemical shown to cause hypolipidemic and peroxisome proliferation, similar to clofibrate, ciprofibrate and DEHP, but structurally different from these three chemicals. Thus, two of the four chemicals are structurally similar while the remaining two are very distinct, although all four chemicals cause the same gross biological response. Our results show that although common protein effects are observed in mice exposed to these chemicals, each chemical also causes specific alterations in selective subsets of proteins that could serve as markers of a particular exposure. 13 refs., 4 figs., 1 tab.

  6. Micropatterning for quantitative analysis of protein-protein interactions in living cells

    E-print Network

    Cai, Long

    the interaction network of molecules in living cells is key to understanding the mechanisms that regulate cell such interaction networks is based on affinity purification of interaction partners of a bait protein and subse. A different approach to sensing molecular proximity is based on energy transfer between a donor

  7. Quantitative Liver-Specific Protein Fingerprint in Blood: A Signature for Hepatotoxicity

    PubMed Central

    Hu, Zhiyuan; Lausted, Christopher; Yoo, Hyuntae; Yan, Xiaowei; Brightman, Amy; Chen, Jiankui; Wang, Weizhi; Bu, Xiangli; Hood, Leroy

    2014-01-01

    We discuss here a new approach to detecting hepatotoxicity by employing concentration changes of liver-specific blood proteins during disease progression. These proteins are capable of assessing the behaviors of their cognate liver biological networks for toxicity or disease perturbations. Blood biomarkers are highly desirable diagnostics as blood is easily accessible and baths virtually all organs. Fifteen liver-specific blood proteins were identified as markers of acetaminophen (APAP)-induced hepatotoxicity using three proteomic technologies: label-free antibody microarrays, quantitative immunoblotting, and targeted iTRAQ mass spectrometry. Liver-specific blood proteins produced a toxicity signature of eleven elevated and four attenuated blood protein levels. These blood protein perturbations begin to provide a systems view of key mechanistic features of APAP-induced liver injury relating to glutathione and S-adenosyl-L-methionine (SAMe) depletion, mitochondrial dysfunction, and liver responses to the stress. Two markers, elevated membrane-bound catechol-O-methyltransferase (MB-COMT) and attenuated retinol binding protein 4 (RBP4), report hepatic injury significantly earlier than the current gold standard liver biomarker, alanine transaminase (ALT). These biomarkers were perturbed prior to onset of irreversible liver injury. Ideal markers should be applicable for both rodent model studies and human clinical trials. Five of these mouse liver-specific blood markers had human orthologs that were also found to be responsive to human hepatotoxicity. This panel of liver-specific proteins has the potential to effectively identify the early toxicity onset, the nature and extent of liver injury and report on some of the APAP-perturbed liver networks. PMID:24465277

  8. Quantitative liver-specific protein fingerprint in blood: a signature for hepatotoxicity.

    PubMed

    Hu, Zhiyuan; Lausted, Christopher; Yoo, Hyuntae; Yan, Xiaowei; Brightman, Amy; Chen, Jiankui; Wang, Weizhi; Bu, Xiangli; Hood, Leroy

    2014-01-01

    We discuss here a new approach to detecting hepatotoxicity by employing concentration changes of liver-specific blood proteins during disease progression. These proteins are capable of assessing the behaviors of their cognate liver biological networks for toxicity or disease perturbations. Blood biomarkers are highly desirable diagnostics as blood is easily accessible and baths virtually all organs. Fifteen liver-specific blood proteins were identified as markers of acetaminophen (APAP)-induced hepatotoxicity using three proteomic technologies: label-free antibody microarrays, quantitative immunoblotting, and targeted iTRAQ mass spectrometry. Liver-specific blood proteins produced a toxicity signature of eleven elevated and four attenuated blood protein levels. These blood protein perturbations begin to provide a systems view of key mechanistic features of APAP-induced liver injury relating to glutathione and S-adenosyl-L-methionine (SAMe) depletion, mitochondrial dysfunction, and liver responses to the stress. Two markers, elevated membrane-bound catechol-O-methyltransferase (MB-COMT) and attenuated retinol binding protein 4 (RBP4), report hepatic injury significantly earlier than the current gold standard liver biomarker, alanine transaminase (ALT). These biomarkers were perturbed prior to onset of irreversible liver injury. Ideal markers should be applicable for both rodent model studies and human clinical trials. Five of these mouse liver-specific blood markers had human orthologs that were also found to be responsive to human hepatotoxicity. This panel of liver-specific proteins has the potential to effectively identify the early toxicity onset, the nature and extent of liver injury and report on some of the APAP-perturbed liver networks. PMID:24465277

  9. Recent advances in preparative electrophoresis

    NASA Technical Reports Server (NTRS)

    Mosher, Richard A.; Thormann, Wolfgang; Egen, Ned B.; Couasnon, Pascal; Sammons, David W.

    1987-01-01

    Various approaches for preparative electrophoresis, and three new instruments for preparative electrophoresis are discussed. Consideration is given to isoelectric focusing, isotachophoresis, and zone electrophoresis, three gel-based electrophoresis methods. The design, functions, and performance of the Elphor VaP 21 device of Hannig (1982), the shear-stabilized BIOSTREAM separator of Thompson (1983), and the recycling isoelectric focusing device are described.

  10. Quantitative proteomic analysis reveals protein expression changes in the murine neuronal secretome during apoptosis.

    PubMed

    Thouvenot, Eric; Urbach, Serge; Vigy, Oana; Séveno, Martial; Galéotti, Nathalie; Nguyen, Geneviève; Bockaert, Joël; Marin, Philippe

    2012-12-21

    Neurodegenerative diseases often lack early and specific diagnostic and prognostic biomarkers. Many studies are focusing on the cerebrospinal fluid (CSF) proteome to identify relevant biomarkers and therapeutic targets for these disorders. An alternative approach consists in comparing proteins secreted by healthy neurons and neurons degenerating by apoptosis, one of the mechanisms underlying neuronal death in neurodegenerative diseases. Here, we adapted the stable isotope labeling by amino acids in cell culture (SILAC) technology to primary cultures of mouse cerebellar granule neurons (CGNs), a well-characterized in vitro model of neuronal apoptosis, in order to identify variations in protein release by neurons during apoptosis. Using two different heavy isotope labels followed by liquid chromatography coupled with Fourier transform tandem mass spectrometry, we directly compared the secretome of apoptotic and surviving CGNs. A total of 1375 proteins were identified in CGN-conditioned media. Among these proteins, 47 were differentially expressed in the supernatants of apoptotic and surviving neurons. About 50% of them have been previously identified in human CSF and some are involved in neuronal death or neuroprotection. This list of apoptosis-regulated proteins should be considered when using targeted quantitative proteomics approaches to characterize or validate CSF biomarkers of neurodegenerative disorders. PMID:23009950

  11. Quantitative Correlation Between the Protein Primary Sequences and Secondary Structures in Spider Dragline Silks

    PubMed Central

    Jenkins, Janelle E.; Creager, Melinda S.; Lewis, Randolph V.; Holland, Gregory P.; Yarger, Jeffery L.

    2009-01-01

    Synthetic spider silk holds great potential for use in various applications spanning medical uses to ultra lightweight armor, however producing synthetic fibers with mechanical properties comparable to natural spider silk has eluded the scientific community. Natural dragline spider silks are commonly made from proteins that contain highly repetitive amino acid motifs, adopting an array of secondary structures. Before further advances can be made in the production of synthetic fibers based on spider silk proteins, it is imperative to know the percentage of each amino acid in the protein that forms a specific secondary structure. Linking these percentages to the primary amino acid sequence of the protein will establish a structural foundation for synthetic silk. In this study, Nuclear Magnetic Resonance (NMR) techniques are used to quantify the percentage of Ala, Gly, and Ser that form both ?-sheet and helical secondary structures. The fraction of these three amino acids and their secondary structure are quantitatively correlated to the primary amino acid sequence for the proteins that comprise major and minor ampullate silk from the Nephila clavipes spider providing a blueprint for synthetic spider silks. PMID:20000730

  12. nature biotechnology volume 26 number 8 august 2008 863 Guidelines for reporting the use of gel electrophoresis

    E-print Network

    Cai, Long

    electrophoresis in proteomics To the editor: We wish to alert your readers to the MIAPE Gel Electrophoresis (MIAPE-dimensional gel electrophoresis in a proteomics experiment. Developed through a joint effort between the gel-GE-compliant submission2. Gel electrophoresis facilitates the separation of protein (or peptide) mixtures, usually

  13. Gel Electrophoresis on a Budget to Dye for

    ERIC Educational Resources Information Center

    Yu, Julie H.

    2010-01-01

    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 $800 for each setup, which is cost prohibitive for most classroom budgets. This article…

  14. Quantitative profiling of the membrane proteome in a halophilic archaeon.

    PubMed

    Bisle, Birgit; Schmidt, Alexander; Scheibe, Burghardt; Klein, Christian; Tebbe, Andreas; Kellermann, Joseph; Siedler, Frank; Pfeiffer, Friedhelm; Lottspeich, Friedrich; Oesterhelt, Dieter

    2006-09-01

    We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotope-coded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small. PMID:16804162

  15. Quantitative Time-course Profiling of Parasite and Host Cell Proteins in the Human Malaria Parasite Plasmodium falciparum*

    PubMed Central

    Foth, Bernardo Javier; Zhang, Neng; Chaal, Balbir Kaur; Sze, Siu Kwan; Preiser, Peter Rainer; Bozdech, Zbynek

    2011-01-01

    Studies of the Plasmodium falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intra-erythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade in which most expressed genes exhibit a single transcriptional peak. Because the biochemical and cellular functions of most genes are mediated by the encoded proteins, understanding the relationship between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. Although studies on other organisms show that <50% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on oligonucleotide microarrays and two-dimensional differential gel electrophoresis protein gels. We find that most proteins are represented by more than one isoform, presumably because of post-translational modifications. Like transcripts, most proteins exhibit cyclic abundance profiles with one peak during the IDC, whereas the presence of functionally related proteins is highly correlated. In contrast, the abundance of most parasite proteins peaks significantly later (median 11 h) than the corresponding transcripts and often decreases slowly in the second half of the IDC. Computational modeling indicates that the considerable and varied incongruence between transcript and protein abundance may largely be caused by the dynamics of translation and protein degradation. Furthermore, we present cyclic abundance profiles also for parasite-associated human proteins and confirm the presence of five human proteins with a potential role in antioxidant defense within the parasites. Together, our data provide fundamental insights into transcript-protein relationships in P. falciparum that are important for the correct interpretation of transcriptional data and that may facilitate the improvement and development of malaria diagnostics and drug therapy. PMID:21558492

  16. Quantitative Proteomics Reveal Distinct Protein Regulations Caused by Aggregatibacter actinomycetemcomitans within Subgingival Biofilms

    PubMed Central

    Bao, Kai; Bostanci, Nagihan; Selevsek, Nathalie; Thurnheer, Thomas; Belibasakis, Georgios N.

    2015-01-01

    Periodontitis is an infectious disease that causes the inflammatory destruction of the tooth-supporting (periodontal) tissues, caused by polymicrobial biofilm communities growing on the tooth surface. Aggressive periodontitis is strongly associated with the presence of Aggregatibacter actinomycetemcomitans in the subgingival biofilms. Nevertheless, whether and how A. actinomycetemcomitans orchestrates molecular changes within the biofilm is unclear. The aim of this work was to decipher the interactions between A. actinomycetemcomitans and other bacterial species in a multi-species biofilm using proteomic analysis. An in vitro 10-species “subgingival” biofilm model, or its derivative that included additionally A. actinomycetemcomitans, were anaerobically cultivated on hydroxyapatite discs for 64 h. When present, A. actinomycetemcomitans formed dense intra-species clumps within the biofilm mass, and did not affect the numbers of the other species in the biofilm. Liquid chromatography-tandem mass spectrometry was used to identify the proteomic content of the biofilm lysate. A total of 3225 and 3352 proteins were identified in the biofilm, in presence or absence of A. actinomycetemcomitans, respectively. Label-free quantitative proteomics revealed that 483 out of the 728 quantified bacterial proteins (excluding those of A. actinomycetemcomitans) were accordingly regulated. Interestingly, all quantified proteins from Prevotella intermedia were up-regulated, and most quantified proteins from Campylobacter rectus, Streptococcus anginosus, and Porphyromonas gingivalis were down-regulated in presence of A. actinomycetemcomitans. Enrichment of Gene Ontology pathway analysis showed that the regulated groups of proteins were responsible primarily for changes in the metabolic rate, the ferric iron-binding, and the 5S RNA binding capacities, on the universal biofilm level. While the presence of A. actinomycetemcomitans did not affect the numeric composition or absolute protein numbers of the other biofilm species, it caused qualitative changes in their overall protein expression profile. These molecular shifts within the biofilm warrant further investigation on their potential impact on its virulence properties, and association with periodontal pathogenesis. PMID:25756960

  17. A Chip-Capillary Hybrid Device for Automated Transfer of Sample Pre-Separated by Capillary Isoelectric Focusing to Parallel Capillary Gel Electrophoresis for Two-Dimensional Protein Separation

    PubMed Central

    Lu, Joann J.; Wang, Shili; Li, Guanbin; Wang, Wei; Pu, Qiaosheng; Liu, Shaorong

    2012-01-01

    In this report, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to re-route the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed. PMID:22830584

  18. A quantitative autoradiographic method for the measurement of local rates of brain protein synthesis

    SciTech Connect

    Dwyer, B.E.; Donatoni, P.; Wasterlain, C.G.

    1982-05-01

    We have developed a new method for measuring local rates of brain protein synthesis in vivo. It combines the intraperitoneal injection of a large dose of low specific activity amino acid with quantitative autoradiography. This method has several advantages: 1) It is ideally suited for young or small animals or where immobilizing an animal is undesirable. 2 The amino acid injection ''floods'' amino acid pools so that errors in estimating precursor specific activity, which is especially important in pathological conditions, are minimized. 3) The method provides for the use of a radioautographic internal standard in which valine incorporation is measured directly. Internal standards from experimental animals correct for tissue protein content and self-absorption of radiation in tissue sections which could vary under experimental conditions.

  19. Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

    PubMed Central

    2012-01-01

    Background Fluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs) for further analysis which is subjective and does not allow for comparing different cells and experimental settings. Results We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp) function is fitted to fluorescence loss (FL) inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP), we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ) disease proteins like mutant huntingtin (mtHtt) can form large aggregates called inclusion bodies (IB’s). The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt) between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and experiments. Conclusions We present two complementary methods for quantitative analysis of FLIP experiments in living cells. They provide spatial maps of exchange dynamics and absolute binding parameters of fluorescent molecules to moving intracellular entities, respectively. Our methods should be of great value for quantitative studies of intracellular transport. PMID:23148417

  20. Nonradioactive monitoring of organic and inorganic solute transport into single Xenopus oocytes by capillary zone electrophoresis.

    PubMed Central

    Nussberger, S; Foret, F; Hebert, S C; Karger, B L; Hediger, M A

    1996-01-01

    Transport of organic and inorganic solutes into and out of cells requires specialized transport proteins. Given a sufficiently sensitive analytical method for measuring cellular solute concentrations, it should be possible to monitor solute transport across the plasma membrane at the level of single cells. We report a capillary zone electrophoresis approach that is generally applicable to monitor solute transport into Xenopus laevis oocytes, requires only nanoliters of sample, and involves no radioactive materials. The sensitivity of capillary electrophoresis with UV detection is typically on the order of 10(-5)-10(-6) M, resulting in the mass detection limits in the low femtomole range. We show that capillary zone electrophoresis serves as a simple technique to measure solute transport into oocytes. Studies of the mammalian oligopeptide transporter PepT1 and the Na(+)- and K(+)-coupled epithelial and neuronal glutamate transporter EAAC1 expressed in oocytes demonstrate that transport of the dipeptide Trp-Gly via PepT1 and transport of Na+ and K+ via EAAC1 across the oocyte plasma membrane can be monitored by measuring intracellular tryptophan absorption and by indirect UV detection of inorganic ions, respectively. The CZE method allowed the simultaneous detection of changes of intracellular Na+ and K+ concentrations in response to EAAC1-mediated Na+ cotransport and K+ countertransport. This is the first report of a capillary zone electrophoresis-based quantitative analysis of intracellular components of a single cell in response to transport activity. Images FIGURE 1 FIGURE 6 PMID:8789117

  1. Lost in centrifugation: accounting for transporter protein losses in quantitative targeted absolute proteomics.

    PubMed

    Harwood, Matthew D; Russell, Matthew R; Neuhoff, Sibylle; Warhurst, Geoffrey; Rostami-Hodjegan, Amin

    2014-10-01

    In drug development, considerable efforts are made to extrapolate from in vitro and preclinical findings to predict human drug disposition by using in vitro-in vivo extrapolation (IVIVE) approaches. Use of IVIVE strategies linked with physiologically based pharmacokinetic (PBPK) modeling is widespread, and regulatory agencies are accepting and occasionally requesting model analysis to support licensing submissions. Recently, there has been a drive to improve PBPK models by characterizing the absolute abundance of enzymes, transporters, and receptors within mammalian tissues and in vitro experimental systems using quantitative targeted absolute proteomics (QTAP). The absolute abundance of proteins relevant to processes governing drug disposition provided by QTAP will enable IVIVE-PBPK to incorporate terms for the abundance of enzymes and transporters in target populations. However, most studies that report absolute abundances of enzymes and transporter proteins do so in enriched membrane fractions so as to increase the abundance per sample, and thus the assay's sensitivity, rather than measuring the expected lower abundance in the more biologically meaningful whole cells or tissues. This communication discusses the balance between protein enrichment and potential loss during the preparation of membrane fractions from whole cells or tissues. Accounting for losses with recovery factors throughout the fractionation procedure provides a means to correct for procedural losses, thereby enabling the scaling of protein abundance from subcellular fractions to whole-cell or organ abundances. PBPK models based on corrected abundances will more closely resemble biological systems and facilitate development of more meaningful IVIVE scaling factors, producing more accurate quantitative predictions of drug disposition. PMID:25061159

  2. Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of the current study was to evaluate the His-tag pr...

  3. Identification of quantitative trait loci (QTL) controlling protein, oil, and five major fatty acids’ contents in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improved seed composition in soybean (Glycine max L. Merr.) for protein and oil quality is one of the major goals of soybean breeders. A group of genes that act as quantitative traits with their effects can alter protein, oil, palmitic, stearic, oleic, linoleic, and linolenic acids percentage in soy...

  4. A Quantitative Proteomics Tool To Identify DNA-Protein Interactions in Primary Cells or Blood.

    PubMed

    Hubner, Nina C; Nguyen, Luan N; Hornig, Nadine C; Stunnenberg, Hendrik G

    2015-02-01

    Interactions between transcription factors and genomic DNA, and in particular their impact on disease and cell fate, have been extensively studied on a global level using techniques based on next-generation sequencing. These approaches, however, do not allow an unbiased study of protein complexes that bind to certain DNA sequences. DNA pulldowns from crude lysates combined with quantitative mass spectrometry were recently introduced to close this gap. Established protocols, however, are restricted to cell lines because they are based on metabolic labeling or require large amounts of material. We introduce a high-throughput-compatible DNA pulldown that combines on-bead digestion with direct dimethyl labeling or label-free protein quantification. We demonstrate that our method can efficiently identify transcription factors binding to their consensus DNA motifs in extracts from primary foreskin fibroblasts and peripheral blood mononuclear cells (PBMCs) freshly isolated from human donors. Nuclear proteomes with absolute quantification of nearly 7000 proteins in K562 cells and PBMCs clearly link differential interactions to differences in protein abundance, hence stressing the importance of selecting relevant cell extracts for any interaction in question. As shown for rs6904029, a SNP highly associated with chronic lymphocytic leukemia, our approach can provide invaluable functional data, for example, through integration with GWAS. PMID:25546135

  5. Distinction of thioredoxin transnitrosylation and denitrosylation target proteins by the ICAT quantitative approach

    PubMed Central

    Wu, Changgong; Parrott, Andrew Myles; Liu, Tong; Jain, Mohit Raja; Yang, Yanfei; Sadoshima, Junichi; Li, Hong

    2012-01-01

    S-Nitrosylation is a reversible PTM for regulating protein function. Thioredoxin-1 (Trx1) catalyzes either transnitrosylation or denitrosylation of specific proteins, depending on the redox status of the cysteines within its conserved oxidoreductase CXXC motif. With a disulfide bond formed between the two catalytic cysteines, Trx1 is not only inactive as a denitrosylase, but it may also be nitrosylated at Cys73 and serve as a transnitrosylating agent. Identification of Trx1-mediated transnitrosylation or denitrosylation targets will contribute to a better understanding of Trx1’s function. Previous experimental approaches based on the attenuation of CXXC oxidoreductase activity cannot readily distinguish Trx1 transnitrosylation targets from denitrosylation targets. In this study, we used the ICAT method in conjunction with the biotin switch technique to differentiate Trx1 transnitrosylation targets from denitrosylation target proteins from neuroblastoma cells. We demonstrate that the ICAT approach is effective for quantitative identification of putative Trx1 transnitrosylation and denitrosylation target peptides. From these analyses, we confirmed reports that peroxiredoxin 1 is a Trx1 transnitrosylation, but not a denitrosylation target, and we found several other proteins, including cyclophilin A to be modulated in this manner. Unexpectedly, we found that many nitrosylation sites are reversibly regulated by Trx1, suggesting a more prominent role for Trx1 in regulating S-nitrosylation. PMID:21704743

  6. Quantitative Characterization of Polymer–Polymer, Protein–Protein, and Polymer–Protein Interaction via Tracer Sedimentation Equilibrium

    PubMed Central

    Fodeke, Adedayo A.; Minton, Allen P.

    2012-01-01

    Quantitative analysis of the composition dependence of the concentration gradient of each species of macromolecule within a solution mixture at sedimentation equilibrium permits the quantitative characterization of self- and heterointeractions between sedimenting solutes. Sedimentation equilibrium experiments were conducted on solutions containing a trace concentration of FITC-labeled BSA in varying concentrations of Ficoll 70 and on solutions containing a trace concentration of FITC-labeled Ficoll 70 in varying concentrations of BSA. The equilibrium gradient of each solute component in each mixture was measured independently. Analysis of the resulting gradients resulted in evaluation of the dependence of the activity coefficient of Ficoll upon the concentrations of Ficoll and BSA at concentrations of up to 100 g/L and the dependence of the activity coefficient of BSA upon the concentrations of Ficoll and BSA at concentrations of up to 100 g/L. The activity coefficients of both species increase significantly with increasing Ficoll and BSA concentration and do not vary with temperature, to within the precision of measurement, over the temperature range of 5–37 °C, indicating that the dominant interaction between Ficoll molecules and between BSA and Ficoll molecules is repulsive and probably due to steric volume exclusion. The measured dependences may be accounted for quantitatively by a simple model in which BSA and Ficoll 70 are represented by equivalent rigid particles. PMID:20677765

  7. ELECTROPHORESIS 08www.electrophoresis-journal.com

    E-print Network

    Xuan, Xiangchun "Schwann"

    with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism and metalloproteins in open tubular capillary electrochromatography with etched chemically modified columns J. J

  8. Quantitative analysis in the characterization and optimization of protein crystal growth.

    PubMed

    Carter, C W; Yin, Y

    1994-07-01

    Protein crystal growth often depends on the combination of many different factors. Some affect protein solubility directly; others may act indirectly by causing conformational changes. Systematic characterization of these factors can be important for generating good crystals. It can also provide useful insight into the biochemical behavior of the protein being crystallized. Here we focus on statistical methods to achieve these two objectives. (1) Characterization of a protein system by analyzing patterns of crystal polymorphism under different levels of biochemical parameters, such as ligands and pH. Tests of the reproducibility of crystal growth experiments indicate that quantitative scales of crystal quality can be statistically significant. Analysis of variance for a replicated, full-factorial design in which four factors were tested at two levels has been used to demonstrate highly significant, biochemically relevant, two-factor interactions strongly implicating pH and ligand-dependent conformational changes. (2) Optimization of crystal growth via response-surface methods. 'Minimum predicted variance' designs provide for efficient response-surface experiments aimed at constructing quadratic models in several dimensions. We have used such models to improve crystal size and quality significantly for three forms of Bacillus stearothermophilus tryptophanyl-tRNA synthetase. In one case we can now avoid having to increase the size by repeated seeding, a difficult procedure that also produces unwanted growth of satellite crystals. Graphs of two-dimensional level surfaces reveal a number of ridges, where the same result is obtained for many combinations of the factors usually varied when trying to improve crystals. An important inference is that it may be better to sample simultaneously for the effects of protein concentration and supersaturation. For a system involving only one crystallizing agent, supersaturation can be approximated as the product of protein and precipitant concentrations. Use of this search direction significantly improves the performance of response-surface experiments. Advantages of growing crystals at stationary points of their response surfaces include better crystals and higher reproducibility, since crystal growth at stationary points is insulated from the deleterious effects of experimental fluctuations. This arises because the derivatives of the response are by definition zero with respect to the experimental variables. Quantitative analysis of appropriately designed crystal growth experiments can thus be a powerful way to characterize complex and interacting biochemical dependencies in macromolecular systems and optimize parameters important to the crystallography. PMID:15299421

  9. Gel Electrophoresis of Dyes

    NSDL National Science Digital Library

    Janice Stephens

    2011-01-01

    In this experiment related to plant biotechnology, learners discover how to prepare and load an electrophoresis gel. They will then run the gels in an electrophoresis system to separate several dyes that are of different molecular sizes and carry different charges. This technique is fundamental to many of the procedures used in biotechnology. This lesson guide includes background information for the educator, safety precautions, and questions with answers for learners. For safety reasons, adult supervision is recommended. Modifications for use with younger learners are described in a related PDF (see related resource).

  10. 2-D gel electrophoresis: constructing 2D-gel proteome reference maps.

    PubMed

    Simula, Maria Paola; Notarpietro, Agata; Toffoli, Giuseppe; De Re, Valli

    2012-01-01

    Two-dimensional gel electrophoresis (2-DE) is the most popular and versatile method of protein separation among a rapidly growing array of proteomic technologies. Based on two independent biochemical characteristics of proteins, it combines isoelectric focusing, which separates proteins according to their isoelectric point (pI), and SDS-PAGE, which separates them further according to their molecular mass. An evolution of conventional 2-DE is represented by the 2D-Difference in Gel Electrophoresis (2D-DIGE) that allows sample multiplexing and achieving more accurate and sensitive quantitative proteomic determinations. The 2-DE separation permits the generation of protein maps of different cells or tissues and the study, by differential proteomics, of protein expression changes associated to the different states of a biological system. In order to identify the molecular bases of pathological processes, it is also useful to characterize the physiological protein homeostasis in healthy cells or tissues. On these grounds, the availability of detailed 2D reference maps could be very useful for proteomic studies. The protocol described in this chapter is based on the 2D-DIGE technology and has been applied to obtain the first 2-DE reference map of the human small intestine. PMID:22130991

  11. A tunable isoelectric focusing via moving reaction boundary for two-dimensional gel electrophoresis and proteomics.

    PubMed

    Guo, Chen-Gang; Shang, Zhi; Yan, Jian; Li, Si; Li, Guo-Qing; Liu, Rong-Zhong; Qing, Ying; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

    2015-05-01

    Routine native immobilized pH gradient isoelectric focusing (IPG-IEF) and two-dimensional gel electrophoresis (2DE) are still suffering from unfortunate reproducibility, poor resolution (caused by protein precipitation) and instability in characterization of intact protein isoforms and posttranslational modifications. Based on the concept of moving reaction boundary (MRB), we firstly proposed a tunable non-IPG-IEF system to address these issues. By choosing proper pairs of catholyte and anolyte, we could achieve desired cathodic and anodic migrating pH gradients in non-IPG-IEF system, effectively eliminating protein precipitation and uncertainty of quantitation existing in routine IEF and 2DE, and enhancing the resolution and sensitivity of IEF. Then, an adjustable 2DE system was developed by combining non-IPG-IEF with polyacrylamide gel electrophoresis (PAGE). The improved 2DE was evaluated by testing model proteins and colon cancer cell lysates. The experiments revealed that (i) a tunable pH gradient could be designed via MRB; (ii) up to 1.65 fold improvement of resolution was achieved via non-IPG-IEF; (iii) the sensitivity of developed techniques was increased up to 2.7 folds; and (iv) up to about 16.4% more protein spots could be observed via the adjustable 2DE as compared with routine one. The developed techniques might contribute to complex proteome research, especially for screening of biological marker and analysis of extreme acidic/alkaline proteins. PMID:25770625

  12. 21 CFR 862.2485 - Electrophoresis apparatus for clinical use.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...for clinical use. (a) Identification. An electrophoresis apparatus for clinical use is a device intended to separate molecules or particles, including plasma proteins, lipoproteins, enzymes, and hemoglobulins on the basis of their net...

  13. 21 CFR 862.2485 - Electrophoresis apparatus for clinical use.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...for clinical use. (a) Identification. An electrophoresis apparatus for clinical use is a device intended to separate molecules or particles, including plasma proteins, lipoproteins, enzymes, and hemoglobulins on the basis of their net...

  14. 21 CFR 862.2485 - Electrophoresis apparatus for clinical use.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...for clinical use. (a) Identification. An electrophoresis apparatus for clinical use is a device intended to separate molecules or particles, including plasma proteins, lipoproteins, enzymes, and hemoglobulins on the basis of their net...

  15. 21 CFR 862.2485 - Electrophoresis apparatus for clinical use.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...for clinical use. (a) Identification. An electrophoresis apparatus for clinical use is a device intended to separate molecules or particles, including plasma proteins, lipoproteins, enzymes, and hemoglobulins on the basis of their net...

  16. Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

    PubMed Central

    Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

    2014-01-01

    Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

  17. Quantitative analysis of immunofluorescent punctate staining of synaptically localized proteins using confocal microscopy and stereology.

    PubMed

    Mokin, Maxim; Keifer, Joyce

    2006-10-30

    We established a protocol for the immunofluorescent detection of glutamate receptor subunits at synaptic sites using laser scanning confocal microscopy and stereological procedures. An in vitro model of eyeblink classical conditioning from turtles was used for this study. Triple-labeling of the presynaptic marker synaptophysin, the NR1 subunit of NMDA receptors, and the GluR4 subunit of AMPA receptors was performed on pseudoconditioned (control) and conditioned in vitro brain stem preparations in which punctate staining for each individual protein, as well as for the colocalization of GluR4 and NR1 with synaptophysin, was analyzed. For every tissue section analyzed, images of two consecutive optical planes were taken using confocal microscopy. Protein puncta were counted in one optical section (sample section) if they were not present in the optical section immediately above the sample section (look-up section). We found a significant increase in the colocalization of GluR4-containing AMPA receptors with synaptophysin after conditioning compared with the control group. Colocalization of NR1 subunits with synaptophysin was unchanged after conditioning. The described protocol, therefore, can be used for the quantitative analysis of changes in synaptic localization of different types of proteins. The protocol is designed to provide a more accurate and uniform approach in studying receptor trafficking during various forms of synaptic plasticity. PMID:16740315

  18. Comparison of quantitative spectral similarity analysis methods for protein higher-order structure confirmation.

    PubMed

    Teska, Brandon M; Li, Cynthia; Winn, Bradley C; Arthur, Kelly K; Jiang, Yijia; Gabrielson, John P

    2013-03-01

    Optical and vibrational spectroscopic techniques are important tools for evaluating secondary and tertiary structures of proteins. These spectroscopic techniques are routinely applied in biopharmaceutical development to elucidate structural characteristics of protein products, to evaluate the impact of processing and storage conditions on product quality, and to assess comparability of a protein product before and after manufacturing changes. Conventionally, the degree of similarity between two spectra has been determined visually. In addition to requiring a significant amount of analyst training and experience, visual inspection of spectra is inherently subjective, and any determination of comparability based on visual analysis of spectra is therefore arbitrary. Here, we discuss a general methodology for evaluating the suitability of numerical methods to calculate spectral similarity, and then we apply the methodology to compare four quantitative spectral similarity methods: the correlation coefficient, area of spectral overlap, derivative correlation algorithm, and spectral difference methods. While the most effective spectral similarity method may depend on the particular application, all four approaches are superior to visual evaluation, and each is suitable for assessing the degree of similarity between spectra. PMID:23219560

  19. Dynamics of Natural Killer Cell Receptor Revealed by Quantitative Analysis of Photoswitchable Protein

    NASA Astrophysics Data System (ADS)

    Pageon, Sophie V.; Aquino, Gerardo; Lagrue, Kathryn; Köhler, Karsten; Endres, Robert G.; Davis, Daniel M.

    2013-11-01

    Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies which rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report a novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, following a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 +- 0.06 micron^2/s) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse and remains surprisingly stable there. Unexpectedly however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected inter-synaptic exchange of protein.

  20. A new fusion protein platform for quantitatively measuring activity of multiple proteases

    PubMed Central

    2014-01-01

    Background Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases. Results We tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract. Conclusion The results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with sufficient substrate specificity. PMID:24649897

  1. Rapid and quantitative detection of C-reactive protein using quantum dots and immunochromatographic test strips

    PubMed Central

    Cheng, Xianglin; Pu, Xu; Jun, Pen; Zhu, XiaoBo; Zhu, Di; Chen, Ming

    2014-01-01

    Background Rapid immunochromatographic tests can detect disease markers in 10–15 minutes, which facilitates clinical diagnosis and treatment programs. However, most immunochromatographic tests employ gold nanoparticles as reporters, and these have only moderate sensitivity and act as qualitative methods for analyzing high biomarker concentrations. Methods In this study, we introduce quantum dots (QDs) as fluorescent probes and immunochromatographic strips to develop quantitative fluorescence point-of-care tests (QF-POCT) to analyze C-reactive protein (CRP) levels. Goat anti-rabbit IgG and rabbit IgG were used as control antibodies, and mouse monoclonal CRP antibody pairs were used for disease marker detection. One monoclonal CRP antibody was conjugated with QDs and served as a signal antibody, and the other monoclonal CRP antibody was dispensed onto the nitrocellulose membrane and served as a capturing antibody. In the presence of CRP, the fluorescence intensity of the monoclonal antibody-CRP-monoclonal antibody sandwich complex captured on the nitrocellulose membrane was determined using the fluorescence strip reader. Results QF-POCT assays could quantitatively analyze the concentration of CRP in 15 minutes had a detection limit of 0.25 mg/L, and had a wide detection linearity range (0.5–300 mg/L). The intra-assay and interassay coefficients of variation were 8.95% and 9.86% at 0.5 mg/L, 6.47% and 8.66% at 10 mg/L, and 6.81% and 9.10% at 60 mg/L, respectively. In a comparison between clinical samples, the results of this QD-based assay of CRP levels were significantly correlated with those of an Immulite 2000 assay (R=0.993, P<0.001). Conclusion Our results demonstrated that the QD-based immunochromatographic test is a rapid, sensitive, accurate, and quantitative method for the detection of disease biomarkers. PMID:25506215

  2. Fraction collector for electrophoresis

    NASA Technical Reports Server (NTRS)

    Bier, M.

    1977-01-01

    Rotating-tube electrophoresis apparatus employs rotating jet of eluting buffer to reduce effects of convection during separation. Designed for separation of microorganisms and biological species, system combines gravity/gradient compensating of lumen with buffer flush at fraction outlet to increase separation efficiency.

  3. DNA ELECTROPHORESIS AT SURFACES

    SciTech Connect

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  4. Chemical methods for the simultaneous quantitation of metabolites and proteins from single cells.

    PubMed

    Xue, Min; Wei, Wei; Su, Yapeng; Kim, Jungwoo; Shin, Young Shik; Mai, Wilson X; Nathanson, David A; Heath, James R

    2015-04-01

    We describe chemical approaches for integrated metabolic and proteomic assays from single cells. Quantitative assays for intracellular metabolites, including glucose uptake and three other species, are designed as surface-competitive binding assays with fluorescence readouts. This enables integration into a microarray format with functional protein immunoassays, all of which are incorporated into the microchambers of a single-cell barcode chip (SCBC). By using the SCBC, we interrogate the response of human-derived glioblastoma cancer cells to epidermal growth factor receptor inhibition. We report, for the first time, on both the intercellular metabolic heterogeneity as well as the baseline and drug-induced changes in the metabolite-phosphoprotein correlation network. PMID:25789560

  5. Methods for quantitative evaluation of dynamics of repair proteins within irradiated cells

    NASA Astrophysics Data System (ADS)

    Hable, V.; Dollinger, G.; Greubel, C.; Hauptner, A.; Krücken, R.; Dietzel, S.; Cremer, T.; Drexler, G. A.; Friedl, A. A.; Löwe, R.

    2006-04-01

    Living HeLa cells are irradiated well directed with single 100 MeV oxygen ions by the superconducting ion microprobe SNAKE, the Superconducting Nanoscope for Applied Nuclear (= Kern-) Physics Experiments, at the Munich 14 MV tandem accelerator. Various proteins, which are involved directly or indirectly in repair processes, accumulate as clusters (so called foci) at DNA-double strand breaks (DSBs) induced by the ions. The spatiotemporal dynamics of these foci built by the phosphorylated histone ?-H2AX are studied. For this purpose cells are irradiated in line patterns. The ?-H2AX is made visible under the fluorescence microscope using immunofluorescence techniques. Quantitative analysis methods are developed to evaluate the data of the microscopic images in order to analyze movement of the foci and their changing size.

  6. Strigolactone-Regulated Proteins Revealed by iTRAQ-Based Quantitative Proteomics in Arabidopsis

    SciTech Connect

    Li, Zhou [ORNL] [ORNL; Czarnecki, Olaf [ORNL] [ORNL; Chourey, Karuna [ORNL] [ORNL; Yang, Jun [ORNL] [ORNL; Tuskan, Gerald A [ORNL] [ORNL; Hurst, Gregory {Greg} B [ORNL; Pan, Chongle [ORNL] [ORNL; Chen, Jay [ORNL] [ORNL

    2014-01-01

    Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. Here, a quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found SLs regulate the expression of about three dozens of proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

  7. A simple and rapid approach for measurement of dissociation constants of DNA aptamers against proteins and small molecules via automated microchip electrophoresis.

    PubMed

    Hu, Jiaming; Easley, Christopher J

    2011-09-01

    Automated microchip electrophoresis was used as a simple and rapid method to measure effective dissociation constants (K(d,eff)) of aptamers against both large and small molecule targets. Human thrombin, immunoglobulin E (IgE), and adenosine triphosphate (ATP) were selected as model analytes to validate the method, with four ligands including two DNA aptamers for thrombin (two distinct epitopes), an IgE aptamer, and an ATP aptamer. The approach is based on a microchip version of a DNA mobility shift assay. Non-denaturing microchip gel electrophoresis separations of DNA could resolve and quantify unbound from target-bound aptamers when using large molecules as targets. To extend the technique to small molecule targets such as ATP, an aptamer/competitor strategy was used, in which a DNA competitor complementary to the aptamer could be displaced by ATP and electrophoretically resolved. Using an automated microchip electrophoresis platform, parallel separations of 11 titration samples were completed in ~0.5 h. Analytical performance comparisons show that our approach provides significant advantages in minimized reagent consumption (typically tens of pmol of aptamer and target), reduced analysis time, and minimized user interaction when compared to previously reported methods for aptamer K(d) measurement. Moreover, the flexibility and ease of K(d,eff) measurement for aptamers against large and small targets make this a unique and valuable approach that should find widespread use. Finally, the feasibility of using this method during aptamer selection processes (e.g. SELEX) was shown by accurate bulk K(d,eff) measurement of a known thrombin aptamer (THRaptA) spiked into a random-sequence DNA pool at as low as 5.0% (molar %) of the total pool; only ~825 fmol of total binding sequences were needed for an 11-point titration curve. PMID:21293790

  8. Thin-layer immunoaffinity chromatography with bar code quantitation of C-reactive protein.

    PubMed

    Nilsson, S; Lager, C; Laurell, T; Birnbaum, S

    1995-09-01

    A rapid thin-layer immunoaffinity chromatographic method for quantitation in serum of an acute phase reactant, C-reactive protein (CRP), which can differentiate between viral and bacteria] infections, is described, where material and reagent costs are minimal. The analysis is based on the "sandwich" assay format using monoclonal antibodies directed against two sites of CRP. One of the antibodies is covalently bound to defined zones on a thin-layer immunoaffinity chromatography membrane, while the other antibody is covalently bound to deeply dyed blue latex particles. After incubation (CRP sample and latex particles), the CRP-latex immunocomplex is allowed to migrate along the immunoaffinity chromatography membrane. In the presence of antigen, a sandwich is formed between the CRP-latex immunocomplex and membrane-bound antibodies, which results in the appearance of blue lines on the membrane. Antibody immobilization on the TLC membrane is made with a redesigned piezoelectric-driven ink-jet printer. The time required for the analysis is less than 10 min. Quantitation is achieved either by counting the lines visually, with scanning reflectometry, or with a modified bar code reader. The limit of detection was estimated in the low femtomolar range using the naked eye as detector. PMID:8779423

  9. Proteomics of the Injured Rat Sciatic Nerve Reveals Protein Expression Dynamics During Regeneration

    Microsoft Academic Search

    Connie R. Jimenez; Floor J. Stam; Ka Wan Li; Yvonne Gouwenberg; Martin P. Hornshaw; Fred De Winter; Joost Verhaagen; August B. Smit

    2004-01-01

    Using proteomics, we investigated the temporal expres- sion profiles of proteins in rat sciatic nerve after experi- mental crush. Extracts of sciatic nerves collected at 5, 10, and 35 days after injury were analyzed by two-dimen- sional gel electrophoresis and quantitative image analy- sis. Of the 1,500 protein spots resolved on each gel, 121 showed significant regulation during at least

  10. Quantitative Proteomics Reveals the Temperature-Dependent Proteins Encoded by a Series of Cluster Genes in Thermoanaerobacter Tengcongensis*

    PubMed Central

    Chen, Zhen; Wen, Bo; Wang, Quanhui; Tong, Wei; Guo, Jiao; Bai, Xue; Zhao, Jingjing; Sun, Yao; Tang, Qi; Lin, Zhilong; Lin, Liang; Liu, Siqi

    2013-01-01

    Comprehensive and quantitative information of the thermophile proteome is an important source for understanding of the survival mechanism under high growth temperature. Thermoanaerobacter tengcongensis (T. tengcongensis), a typical anaerobic thermophilic eubacterium, was selected to quantitatively evaluate its protein abundance changes in response to four different temperatures. With optimized procedures of isobaric tags for relative and absolute quantitation quantitative proteomics (iTRAQ), such as peptide fractionation with high-pH reverse phase (RP) high performance liquid chromatography (HPLC), tandem MS acquisition mode in LTQ Orbitrap Velos MS, and evaluation of the quantification algorithms, high quality of the quantitative information of the peptides identified were acquired. In total, 1589 unique proteins were identified and defined 251 as the temperature-dependent proteins. Analysis of genomic locations toward the correspondent genes of these temperature-dependent proteins revealed that more than 30% were contiguous units with relevant biological functions, which are likely to form the operon structures in T. tengcongensis. The RNA sequencing (RNA-seq) data further demonstrated that these cluster genes were cotranscribed, and their mRNA abundance changes responding to temperature exhibited the similar trends as the proteomic results, suggesting that the temperature-dependent proteins are highly associated with the correspondent transcription status. Hence, the operon regulation is likely an energy-efficient mode for T. tengcongensis survival. In addition, evaluation to the functions of differential proteomes indicated that the abundance of the proteins participating in sulfur-respiration on the plasma membrane was decreased as the temperature increased, whereas the glycolysis-related protein abundance was increased. The energy supply in T. tengcongensis at high temperature is, therefore, speculated not mainly through the respiration chain reactions. PMID:23665590

  11. A Slot Blot Immunoassay for Quantitative Detection of Plasmodium falciparum Circumsporozoite Protein in Mosquito Midgut Oocyst

    PubMed Central

    Kumar, Sanjai; Zheng, Hong; Deng, Bingbing; Mahajan, Babita; Grabias, Bryan; Kozakai, Yukiko; Morin, Merribeth J.; Locke, Emily; Birkett, Ashley; Miura, Kazutoyo; Long, Carole

    2014-01-01

    There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB) for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP) and native PfCSP from Oocysts (Pf Oocyst) developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5–20 pg; R2?=?0.9505). We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1–4, R2?=?0.9795) and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5–3 pg) of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV) for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes and transmission-blocking interventions in endemic areas. PMID:25531543

  12. A New Strategy of Using O18-Labeled Iodoacetic Acid for Mass Spectrometry-Based Protein Quantitation

    NASA Astrophysics Data System (ADS)

    Wang, Shunhai; Kaltashov, Igor A.

    2012-07-01

    A new O18 labeling protocol is designed to assist quantitation of cysteine-containing proteins using LC/MS. Unlike other O18 labeling strategies, the labeling is carried out at the intact protein level (prior to its digestion) during reduction/alkylation of cysteine side chains using O18-labeled iodoacetic acid (IAA). The latter can be easily prepared by exchanging carboxylic oxygen atoms of commercially available IAA in O18-enriched water at low pH. Since incorporation of the O18 label in the protein occurs at the whole protein, rather than peptide level, the quantitation results are not peptide-dependent. The excellent stability of the label in mild pH conditions provides flexibility and robustness needed of sample processing steps following the labeling. In contrast to generally costly isotope labeling reagents, this approach uses only two relatively inexpensive commercially available reagents (IAA and H2O18). The feasibility of the new method is demonstrated using an 80 kDa human serum transferrin (hTf) as a model, where linear quantitation is achieved across a dynamic range spanning three orders of magnitude. The new approach can be used in quantitative proteomics applications and is particularly suitable for a variety of tasks in the biopharmaceutical sector, ranging from pharmacokinetic studies to quality control of protein therapeutics.

  13. Electrophoresis. Author manuscript Alterations of the mitochondrial proteome caused by the absence of

    E-print Network

    Paris-Sud XI, Université de

    Proteins ; deficiency ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; methods ; Humans is synthetized in mitochondria, which are also a major cellular store for calcium. Important catabolic pathways

  14. Comparison of protein patterns of xrs-5, a radiosensitive Chinese hamster ovary cell line, and CHO-K1, its radioresistant parent, using two-dimensional gel-electrophoresis

    SciTech Connect

    Kramer, J.M. (Miami Univ., Oxford, OH (USA). Dept. of Zoology)

    1991-01-01

    X-ray sensitive strains of Chinese hamster ovary cell lines have been used to analyze radiation repair mechanisms. One cell line, xrs-5, has been shown to be very sensitive to ionizing radiation and radical forming chemical mutagens. This sensitivity is thought to be a result a mutation in the DNA double strand break (DSB) repair mechanism, and its characterization has been a goal of several repair mechanism studies. Using two-dimensional gel electrophoresis, we have detected a protein (MW approximately 55KD) in the DNA/Nuclear Matrix (nucleoid) cell fraction of CHO-Kl cells that is absent in the nucleoid fraction of xrs-5. This protein is present, however, in both CHO-Kl and xrs-5 whole cell protein maps. To determine whether the 55KD protein is responsible for the radiosensitive and defective DSB repair phenotype of xrs-5 cells, studies are now underway to analyze revertants of xrs-5 that are proficient in DSB repair. Furthermore, an effort to sequence the protein in question is planned. 23 refs., 2 figs.

  15. Bayesian Analysis of High-Throughput Quantitative Measurement of Protein-DNA Interactions

    PubMed Central

    Pollock, David D.; de Koning, A. P. Jason; Kim, Hyunmin; Castoe, Todd A.; Churchill, Mair E. A.; Kechris, Katerina J.

    2011-01-01

    Transcriptional regulation depends upon the binding of transcription factor (TF) proteins to DNA in a sequence-dependent manner. Although many experimental methods address the interaction between DNA and proteins, they generally do not comprehensively and accurately assess the full binding repertoire (the complete set of sequences that might be bound with at least moderate strength). Here, we develop and evaluate through simulation an experimental approach that allows simultaneous high-throughput quantitative analysis of TF binding affinity to thousands of potential DNA ligands. Tens of thousands of putative binding targets can be mixed with a TF, and both the pre-bound and bound target pools sequenced. A hierarchical Bayesian Markov chain Monte Carlo approach determines posterior estimates for the dissociation constants, sequence-specific binding energies, and free TF concentrations. A unique feature of our approach is that dissociation constants are jointly estimated from their inferred degree of binding and from a model of binding energetics, depending on how many sequence reads are available and the explanatory power of the energy model. Careful experimental design is necessary to obtain accurate results over a wide range of dissociation constants. This approach, which we call Simultaneous Ultra high-throughput Ligand Dissociation EXperiment (SULDEX), is theoretically capable of rapid and accurate elucidation of an entire TF-binding repertoire. PMID:22069446

  16. Identification of Protein Network Alterations upon Retinal Ischemia-Reperfusion Injury by Quantitative Proteomics Using a Rattus norvegicus Model

    PubMed Central

    Tian, Han; Wang, Leilei; Cai, Ruiqi; Zheng, Ling; Guo, Lin

    2014-01-01

    Retinal ischemia is a common feature associated with several ocular diseases, including diabetic retinopathy. In this study, we investigated the effect of a retinal ischemia and reperfusion (I/R) injury on protein levels via a quantitative shotgun strategy using stable isotope dimethyl labeling combined with LC-MS/MS analysis. Based on the relative quantitation data of 1088 proteins, 234 proteins showed a greater than 1.5-fold change following I/R injury, 194 of which were up-regulated and 40 were down-regulated. Gene ontology analysis revealed that after I/R injury, there was an increase in the metabolic-process related proteins but a decline in cell communication, system process and transport-related proteins. A ribosome protein network and a secreted protein network consisting of many protease inhibitors were identified among the up-regulated proteins, despite a suppression of the mammalian target of rapamycin (mTOR) pathway following the I/R injury. A synaptic-related protein network was found to be significantly down-regulated, implicating a functional reduction of neurons following a retinal I/R injury. Our results provide new systems-biology clues for the study of retinal ischemia. PMID:25549249

  17. Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics

    PubMed Central

    Klein, Marlise I.; Xiao, Jin; Lu, Bingwen; Delahunty, Claire M.; Yates, John R.; Koo, Hyun

    2012-01-01

    Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (P<0.05). Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other organisms. Our data provide insights about how S. mutans optimizes its metabolism and adapts/survives within the mixed-species community in response to a dynamically changing environment. This reflects the intricate physiological processes linked to expression of virulence by this bacterium within complex biofilms. PMID:23049864

  18. Analysis of electrophoresis performance

    NASA Technical Reports Server (NTRS)

    Roberts, G. O.

    1984-01-01

    The SAMPLE computer code models electrophoresis separation in a wide range of conditions. Results are included for steady three dimensional continuous flow electrophoresis (CFE), time dependent gel and acetate film experiments in one or two dimensions and isoelectric focusing in one dimension. The code evolves N two dimensional radical concentration distributions in time, or distance down a CFE chamber. For each time or distance increment, there are six stages, successively obtaining the pH distribution, the corresponding degrees of ionization for each radical, the conductivity, the electric field and current distribution, and the flux components in each direction for each separate radical. The final stage is to update the radical concentrations. The model formulation for ion motion in an electric field ignores activity effects, and is valid only for low concentrations; for larger concentrations the conductivity is, therefore, also invalid.

  19. Happy bicentennial, electrophoresis!

    PubMed

    Righetti, Pier Giorgio

    2009-12-01

    A short survey of electrophoresis and a celebration of its bicentennial, with some remarkable mementos and a list of books that shaped the field. Where one also learns of a secret production plant with a huge-scale electrophoretic apparatus for skimming of latex from Hevea brasiliensis and keeping the wheels of the Ally Army running during World War II. And of cyber (mammoth) 2D gels of 1.5 x 1 m in size accommodating >12,000 spots. PMID:19938305

  20. Quantitative Organelle Proteomics of MCF-7 Breast Cancer Cells Reveals Multiple Subcellular Locations for Proteins in Cellular Functional Processes

    PubMed Central

    Qattan, Amal T.; Mulvey, Claire; Crawford, Mark; Natale, Darren A.; Godovac-Zimmermann, Jasminka

    2014-01-01

    We have combined sucrose density gradient subcellular fractionation with quantitative, tandem-mass-spectrometry-based shotgun proteomics to investigate spatial distributions of proteins in MCF-7 breast cancer cells. Emphasis was placed on four major organellar compartments: cytosol, plasma membrane, endoplasmic reticulum, and mitochondrion. Two-thousand one-hundred eighty-four proteins were securely identified. Four-hundred eighty-one proteins (22.0% of total proteins identified) were found in unique sucrose gradient fractions, suggesting they may have unique subcellular locations. 454 proteins (20.8%) were found to be ubiquitously distributed. The remaining 1249 proteins (57.2%) were consistent with intermediate distribution over multiple, but not all, subcellular locations. Ninety-four proteins implicated in breast cancer and 478 other proteins which share the same five major cellular biological processes with a majority of the breast cancer proteins were observed in 334 and 1223 subcellular locations, respectively. The data obtained is used to evaluate the possibility of defining more exact sets of subcellular organelles, the completeness of current descriptions of spatial distribution of cellular proteins, the importance of multiple subcellular locations for proteins in functional processes, the subcellular distribution of proteins related to breast cancer, and the possibility of using these methods for dynamic spatio/temporal studies of function/regulation in MCF-7 breast cancer cells. PMID:19911851

  1. Quantitative proteomic analysis reveals proteins involved in the neurotoxicity of marine medaka Oryzias melastigma chronically exposed to inorganic mercury.

    PubMed

    Wang, Yuyu; Wang, Dazhi; Lin, Lin; Wang, Minghua

    2015-01-01

    Mercury is a ubiquitous environmental contaminant which exerts neurotoxicity upon animals. Nevertheless, the molecular mechanisms involved in inorganic mercury neurotoxicity are unknown. We investigated protein profiles of marine medaka, chronically exposed to mercuric chloride using two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS) analysis. The mercury accumulation and ultrastructure were also examined in the brain. The results showed that mercury was significantly accumulated in the treated brain, and subsequently caused a noticeable damage. The comparison of 2D-DIGE protein profiles between the control and treatment revealed that 16 protein spots were remarkably altered in abundance, which were further submitted for MALDI-TOF-TOF MS analysis. The identified proteins indicated that inorganic mercury may cause neurotoxicity through the induction of oxidative stress, cytoskeletal assembly dysfunction and metabolic disorders. Thus, this study provided a basis for a better understanding of the molecular mechanisms involved in mercury neurotoxicity. PMID:25460752

  2. Preparative electrophoresis for space

    NASA Technical Reports Server (NTRS)

    Rhodes, Percy H.; Snyder, Robert S.

    1988-01-01

    A premise of continuous flow electrophoresis is that removal of buoyance-induced thermal convection caused by axial and lateral temperature gradients results in ideal performance of these instruments in space. Although these gravity dependent phenomena disturb the rectilinear flow in the separation chamber when high voltage gradients or thick chamber are used, distortion of the injected sample stream due to electrodynamic effects cause major broadening of the separated bands. The electrophoresis separation process is simple, however flow local to the sample filament produced by the applied electric field were not considered. These electrohydrodynamic flows distort the sample stream and limit the separation. Also, electroosmosis and viscous flow combine to further distort the process. A moving wall concept is being proposed for space which will eliminate and control the disturbances. The moving wall entrains the fluid to move as a rigid body and produces a constant residence time for all samples distributed across the chamber thickness. The moving wall electrophoresis chamber can only be operated in space because there is no viscous flow in the chamber to stabilize against thermal convection.

  3. Preparative electrophoresis for space

    NASA Technical Reports Server (NTRS)

    Rhodes, Percy H.; Snyder, Robert S.

    1987-01-01

    A premise of continuous flow electrophoresis is that removal of buoyancy-induced thermal convection caused by axial and lateral temperature gradients results in ideal performance of these instruments in space. Although these gravity dependent phenomena disturb the rectilinear flow in the separation chamber when high voltage gradients or thick chambers are used, distortion of the injected sample stream due to electrohydrodynamic effects cause major broadening of the separated bands. The electrophoresis separation process is simple, however flow local to the sample filament produced by the applied electric field have not been considered. These electrohydrodynamic flows distort the sample stream and limit the separation. Also, electroosmosis and viscous flow combine to further distort the process. A moving wall concept is being proposed for space which will eliminate and control the disturbances. The moving wall entrains the fluid to move as a rigid body and produces a constant residence time for all samples distributed across the chamber thickness. The moving wall electrophoresis chamber can only be operated in space because there is no viscous flow in the chamber to stabilize against thermal convection.

  4. Genetics Coupled to Quantitative Intact Proteomics Links Heritable Aphid and Endosymbiont Protein Expression to Circulative Polerovirus Transmission? †

    PubMed Central

    Cilia, M.; Tamborindeguy, C.; Fish, T.; Howe, K.; Thannhauser, T. W.; Gray, S.

    2011-01-01

    Yellow dwarf viruses in the family Luteoviridae, which are the causal agents of yellow dwarf disease in cereal crops, are each transmitted most efficiently by different species of aphids in a circulative manner that requires the virus to interact with a multitude of aphid proteins. Aphid proteins differentially expressed in F2 Schizaphis graminum genotypes segregating for the ability to transmit Cereal yellow dwarf virus-RPV (CYDV-RPV) were identified using two-dimensional difference gel electrophoresis (DIGE) coupled to either matrix-assisted laser desorption ionization-tandem mass spectrometry or online nanoscale liquid chromatography coupled to electrospray tandem mass spectrometry. A total of 50 protein spots, containing aphid proteins and proteins from the aphid's obligate and maternally inherited bacterial endosymbiont, Buchnera, were identified as differentially expressed between transmission-competent and refractive aphids. Surprisingly, in virus transmission-competent F2 genotypes, the isoelectric points of the Buchnera proteins did not match those in the maternal Buchnera proteome as expected, but instead they aligned with the Buchnera proteome of the transmission-competent paternal parent. Among the aphid proteins identified, many were involved in energy metabolism, membrane trafficking, lipid signaling, and the cytoskeleton. At least eight aphid proteins were expressed as heritable, isoelectric point isoform pairs, one derived from each parental lineage. In the F2 genotypes, the expression of aphid protein isoforms derived from the competent parental lineage aligned with the virus transmission phenotype with high precision. Thus, these isoforms are candidate biomarkers for CYDV-RPV transmission in S. graminum. Our combined genetic and DIGE approach also made it possible to predict where several of the proteins may be expressed in refractive aphids with different barriers to transmission. Twelve proteins were predicted to act in the hindgut of the aphid, while six proteins were predicted to be associated with the accessory salivary glands or hemolymph. Knowledge of the proteins that regulate virus transmission and their predicted locations will aid in understanding the biochemical mechanisms regulating circulative virus transmission in aphids, as well as in identifying new targets to block transmission. PMID:21159868

  5. Genetics coupled to quantitative intact proteomics links heritable aphid and endosymbiont protein expression to circulative polerovirus transmission.

    PubMed

    Cilia, M; Tamborindeguy, C; Fish, T; Howe, K; Thannhauser, T W; Gray, S

    2011-03-01

    Yellow dwarf viruses in the family Luteoviridae, which are the causal agents of yellow dwarf disease in cereal crops, are each transmitted most efficiently by different species of aphids in a circulative manner that requires the virus to interact with a multitude of aphid proteins. Aphid proteins differentially expressed in F2 Schizaphis graminum genotypes segregating for the ability to transmit Cereal yellow dwarf virus-RPV (CYDV-RPV) were identified using two-dimensional difference gel electrophoresis (DIGE) coupled to either matrix-assisted laser desorption ionization-tandem mass spectrometry or online nanoscale liquid chromatography coupled to electrospray tandem mass spectrometry. A total of 50 protein spots, containing aphid proteins and proteins from the aphid's obligate and maternally inherited bacterial endosymbiont, Buchnera, were identified as differentially expressed between transmission-competent and refractive aphids. Surprisingly, in virus transmission-competent F2 genotypes, the isoelectric points of the Buchnera proteins did not match those in the maternal Buchnera proteome as expected, but instead they aligned with the Buchnera proteome of the transmission-competent paternal parent. Among the aphid proteins identified, many were involved in energy metabolism, membrane trafficking, lipid signaling, and the cytoskeleton. At least eight aphid proteins were expressed as heritable, isoelectric point isoform pairs, one derived from each parental lineage. In the F2 genotypes, the expression of aphid protein isoforms derived from the competent parental lineage aligned with the virus transmission phenotype with high precision. Thus, these isoforms are candidate biomarkers for CYDV-RPV transmission in S. graminum. Our combined genetic and DIGE approach also made it possible to predict where several of the proteins may be expressed in refractive aphids with different barriers to transmission. Twelve proteins were predicted to act in the hindgut of the aphid, while six proteins were predicted to be associated with the accessory salivary glands or hemolymph. Knowledge of the proteins that regulate virus transmission and their predicted locations will aid in understanding the biochemical mechanisms regulating circulative virus transmission in aphids, as well as in identifying new targets to block transmission. PMID:21159868

  6. Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF

    EPA Science Inventory

    Introduction: Differential protein expression studies have been routinely performed in our laboratory to determine the health effects of environmentally-important chemicals. In this abstract, improvements in the in-gel protein digestion, MALDI plate spotting and data acquisition...

  7. A chemiluminescent-western blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein.

    PubMed

    Kumar, Sanjai; Zheng, Hong; Sangweme, Davison T; Mahajan, Babita; Kozakai, Yukiko; Pham, Phuong T; Morin, Merribeth J; Locke, Emily; Kumar, Nirbhay

    2013-04-30

    Highly sensitive and reliable assays based on the quantitation of immunologically relevant component(s) in recombinant or whole parasite-based vaccines would facilitate pre-clinical and clinical phases and the monitoring of malaria vaccine deployment. Here we report a laboratory-grade Western Blot assay for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in P. falciparum sporozoite (PfSPZ) and in recombinant (rPfCSP) product. This assay is based on the immuno-reactivity of an anti-P. falciparum CSP monoclonal antibody (mAb 2A10) with the NANP-repeat units on PfCSP. The antigen-antibody complex is detected by reaction with a commercially obtained chemiluminescence-linked Immunodetection system. The linear range for detecting the recombinant P. falciparum CSP (rPfCSP) in this assay is 3-12pg (R(2)=0.9399). The range for detecting the day 15 salivary-gland PfSPZ is between 0.0625 and 1 parasite (R(2)=0.9448) and approximately 10.0pg of PfCSP was detected on each sporozoite. The assay was highly reproducible in measuring the PfCSP on PfSPZ. The inter-assay Coefficient of Variation (CV%) was 10.31% while the intra-assay CV% on three different days was 6.05%, 2.03% and 1.42% respectively. These results suggest that this ECL-WB assay is highly sensitive and robust with a low degree of inter-assay and intra-assay variations. To our knowledge, this is the most sensitive immunoassay for the detection of a recombinant or native malarial protein and may have a wider range of applications including the quantification of immunological component(s) in a vaccine formulation, determination of the antigenic integrity in adjuvanted-vaccine and in stability studies. In addition, this assay can be applied to measure the mosquito infectivity in malaria transmission areas and to determine the effects of intervention measures on malaria transmission. PMID:23399449

  8. Applications of space-electrophoresis in medicine. [for cellular separations in molecular biology

    NASA Technical Reports Server (NTRS)

    Bier, M.

    1976-01-01

    The nature of electrophoresis is reviewed and potential advances realizable in the field of biology and medicine from a space electrophoresis facility are examined. The ground-based applications of electrophoresis: (1) characterization of an ionized species; (2) determination of the quantitative composition of a complex mixture; and (3) isolation of the components of a mixture, separation achieved on the basis of the difference in transport rates is reviewed. The electrophoresis of living cells is considered, touching upon the following areas: the separation of T and B lymphocytes; the genetic influence on mouse lymphocyte mobilities; the abnormal production of specific and monoclonal immunoproteins; and the study of cancer. Schematic diagrams are presented of three types of electrophoresis apparatus: the column assembly for the static electrophoresis experiment on the Apollo-Soyuz mission, the continuous flow apparatus used in the same mission and a miniaturized electrophoresis apparatus.

  9. Analysis of proteins using DIGE and MALDI mass spectrometry

    EPA Science Inventory

    In this work the sensitivity of the quantitative proteomics approach 2D-DIGE/MS (twoDimensional Difference Gel Electrophoresis / Mass Spectrometry) was tested by detecting decreasing amounts of a specific protein at the low picomole and sub-picomole range. Sensitivity of the 2D-D...

  10. Combining Capillary Electrophoresis with Mass Spectrometry for Applications in Proteomics

    SciTech Connect

    Simpson, David C.; Smith, Richard D.

    2005-04-01

    Throughout the field of global proteomics, ranging from simple organism studies to human medical applications, the high sample complexity creates demands for improved separations and analysis techniques. Furthermore, with increased organism complexity, the correlation between proteome and genome becomes less certain due to extensive mRNA processing prior to translation. In this way, the same DNA sequence can potentially code for regions in a number of distinct proteins; quantitative differences in expression (or abundance) between these often-related species are of significant interest. Well-established proteomics techniques, which use genomic information to identify peptides that originate from protease digestion, often cannot easily distinguish between such gene products; intact protein-level analyses are required to complete the picture, particularly for identifying post-translational modifications. While chromatographic techniques are currently better suited to peptide analysis, capillary electrophoresis (CE) in combination with mass spectrometry (MS) may become important for intact protein analysis. This review focuses on CE/MS instrumentation and techniques showing promise for such applications, highlighting those with greatest potential. Reference will also be made to developments relevant to peptide-level analyses for use in time- or sample-limited situations.

  11. The effects of shared peptides on protein quantitation in label-free proteomics by LC/MS/MS

    SciTech Connect

    Jin, Shuangshuang; Daly, Don S.; Springer, David L.; Miller, John H.

    2008-01-02

    Assessment of differential protein abundance from the observed properties of detected peptides is an essential part of protein profiling based on shotgun proteomics. However, the abundance observed for degenerate peptides may be due to contributions from multiple proteins that are affected differently by a given treatment. Excluding degenerate peptides eliminates this ambiguity but may significantly decrease the number of proteins for which abundance estimates can be obtained. Peptide degeneracy within a family of biologically related proteins does not cause ambiguity if family members have a common response to treatment. Based on this concept, we have developed an approach for including degenerate peptides in the analysis of differential protein abundance in protein profiling. Data from a recent proteomics study of lung tissue from mice exposed to lipopolysaccharide, cigarette smoke, and a combination of these agents is used to illustrate our method. Starting from data where about half of the protein identifications involved degenerate peptides, 82% of the affected proteins were grouped into families, based on FASTA annotation, with closure on peptide degeneracy. In many cases, a common abundance relative to control was sufficient to explain ion-current peak areas for peptides, both unique and degenerate, that identified biologically-related proteins in a peptide-degeneracy closure group. Based on these results, we propose that peptide-degeneracy closure groups provide a way to include abundance data for degenerate-peptides in quantitative protein profiling by high throughput mass spectrometry.

  12. Using a Label Free Quantitative Proteomics Approach to Identify Changes in Protein Abundance in Multidrug-Resistant Mycobacterium tuberculosis.

    PubMed

    Phong, Truong Quoc; Ha, Do Thi Thu; Volker, Uwe; Hammer, Elke

    2015-06-01

    Reports in recent years indicate that the increasing emergence of resistance to drugs be using to TB treatment. The resistance to them severely affects to options for effective treatment. The emergence of multidrug-resistant tuberculosis has increased interest in understanding the mechanism of drug resistance in M. tuberculosis and the development of new therapeutics, diagnostics and vaccines. In this study, a label-free quantitative proteomics approach has been used to analyze proteome of multidrug-resistant and susceptible clinical isolates of M. tuberculosis and identify differences in protein abundance between the two groups. With this approach, we were able to identify a total of 1,583 proteins. The majority of identified proteins have predicted roles in lipid metabolism, intermediary metabolism, cell wall and cell processes. Comparative analysis revealed that 68 proteins identified by at least two peptides showed significant differences of at least twofolds in relative abundance between two groups. In all protein differences, the increase of some considering proteins such as NADH dehydrogenase, probable aldehyde dehydrogenase, cyclopropane mycolic acid synthase 3, probable arabinosyltransferase A, putative lipoprotein, uncharacterized oxidoreductase and six membrane proteins in resistant isolates might be involved in the drug resistance and to be potential diagnostic protein targets. The decrease in abundance of proteins related to secretion system and immunogenicity (ESAT-6-like proteins, ESX-1 secretion system associated proteins, O-antigen export system and MPT63) in the multidrug-resistant strains can be a defensive mechanism undertaken by the resistant cell. PMID:25805910

  13. Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition

    PubMed Central

    Kronja, Iva; Whitfield, Zachary J.; Yuan, Bingbing; Dzeyk, Kristina; Kirkpatrick, Joanna; Krijgsveld, Jeroen; Orr-Weaver, Terry L.

    2014-01-01

    The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, we previously described proteome remodeling during Drosophila egg activation. Here, we describe our quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey, to our knowledge, of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. We show that Muskelin, found to be up-regulated during oocyte maturation, is required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. We find down-regulation of this class of proteins to be mediated partially by APC/CCORT, a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C). PMID:25349405

  14. Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition.

    PubMed

    Kronja, Iva; Whitfield, Zachary J; Yuan, Bingbing; Dzeyk, Kristina; Kirkpatrick, Joanna; Krijgsveld, Jeroen; Orr-Weaver, Terry L

    2014-11-11

    The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, we previously described proteome remodeling during Drosophila egg activation. Here, we describe our quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey, to our knowledge, of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. We show that Muskelin, found to be up-regulated during oocyte maturation, is required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. We find down-regulation of this class of proteins to be mediated partially by APC/C(CORT), a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C). PMID:25349405

  15. A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

    PubMed Central

    Helka, Blake-Joseph; Brennan, John D.

    2013-01-01

    Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays. PMID:24022739

  16. Characterization of the Human Adipocyte Proteome and Reproducibility of Protein Abundance by One-dimensional Gel Electrophoresis and HPLC-ESI-MS/MS

    PubMed Central

    Xie, Xitao; Yi, Zhengping; Bowen, Benjamin; Wolf, Cassandra; Flynn, Charles R.; Sinha, Sandeep; Mandarino, Lawrence J.; Meyer, Christian

    2010-01-01

    Abnormalities in adipocytes play an important role in various conditions, including the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease, but little is known about alterations at the protein level. We therefore sought to 1) comprehensively characterize the human adipocyte proteome for the first time, and 2) demonstrate feasibility of measuring adipocyte protein abundances by one-dimensional SDS-PAGE and High Performance Liquid Chromatography -Electron Spray Ionization - tandem Mass Spectrometry (HPLC-ESI-MS/MS). In adipocytes isolated from ~0.5 g subcutaneous abdominal adipose tissue of three healthy, lean subjects we identified a total of 1493 proteins. Triplicate analysis indicated a 22.5% coefficient of variation of protein abundances. Proteins ranged from 5.8 to 629 kDa and included a large number of proteins involved in lipid metabolism, such as fatty acid transport, fatty acid oxidation, lipid storage, lipolysis and lipid droplet maintenance. Furthermore, we found most glycolysis enzymes and numerous proteins associated with oxidative stress, protein synthesis and degradation as well as some adipokines. 22% of all proteins were of mitochondrial origin. These results provide the first detailed characterization of the human adipocyte proteome, suggest an important role of adipocyte mitochondria, and demonstrate feasibility of this approach to examine alterations of adipocyte protein abundances in human diseases. PMID:20812759

  17. Targeted quantitative mass spectrometric identification of differentially expressed proteins between Bax-expressing and deficient colorectal carcinoma cells.

    PubMed

    Wang, Peng; Lo, Andy; Young, J Bryce; Song, Jin H; Lai, Raymond; Kneteman, Norman M; Hao, Chunhai; Li, Liang

    2009-07-01

    Bax, a Bcl-2 interacting protein, plays a central role in several stimuli-induced apoptosis pathways through its functional and physical interactions with various biologically important proteins. Identification of the Bax-modulating protein network should be useful to further our understanding of Bax-mediated apoptosis. For the first time, we performed proteome-wide quantification and identification of differentially expressed proteins between Bax+/- and Bax-/- HCT116 clones using a newly developed quantitative mass spectrometric analysis strategy. This strategy is based on forward and reverse differential isotope labeling of the proteome digests of two comparative cells, followed by two-dimensional liquid chromatography separation and automated peptide deposition to matrix-assisted laser desorption ionization sample plates for MS quantification and MS/MS peptide sequence identification. We quantified and identified 200 differentially expressed proteins involved in various cellular processes. Through bioinformatic analysis, four groups of differentially expressed proteins were highlighted for the association with Bax: mitochondria permeability transition channel proteins, Bax regulator proteins, heat shock protein family members, and oxidative stress-triggered proteins. These results indicate the functional diversity of Bax and provide new research directions to study the biology of Bax-regulated apoptosis. PMID:19425606

  18. Proteins Extracted by Water or Aqueous Ethanol During Refining of Developed Wheat Dough to Vital Wheat Gluten and Crude Starch as Determined by Capillary-Zone Electrophoresis (CZE)

    Microsoft Academic Search

    G. H. Robertson; T. K. Cao

    2004-01-01

    Cereal Chem. 81(5):673-680 Fluids applied to large-sale, technical separation of wheat starch and protein also extract soluble proteins. The degree and rate of extraction and the specific components extracted depend on the flour, the flour hydration and development, the starch-displacing fluid composition, the temper- ature, and the mechanical processing method. This study sought to identify major extracted protein groups using

  19. Exploitation of detergent thermodynamics in the direct solubilization of myelin membrane proteins for two-dimensional gel electrophoresis for proteomic analysis.

    PubMed

    Nair, Sreepriya; Xavier, Tessy; Kumar, Madathiparambil Kumaran Satheesh; Saha, Sharmistha; Menon, Krishnakumar N

    2011-12-01

    Performing 2-DE of lipid-rich multilamellar membranes like myelin is a cumbersome task. However, for understanding its molecular organization and changes during diseases, identification of proteins of myelin is essential. Although the 2-D-proteomic approach of myelin has been employed to understand the myelin proteome, representation of myelin proteins in its entirety is still a challenge. 2-DE profiling of myelin proteins is very important for the detection of immuno-reactivity to myelin proteins from various biological fluids following Western blotting in diseases like multiple sclerosis. Here we developed a novel approach by exploiting the thermodynamic principles behind detergent-mediated solubilization of myelin membranes without any conventional processing of myelin involving precipitation of myelin proteins. We show that the addition of myelin to ASB-14-4 resulted in significant increase in protein representation of myelin in 2-DE compared with the addition of ASB-14-4 to myelin. Moreover, the number and resolution of spots are significantly higher in myelin to ASB-14-4 strategy than other strategies of myelin sample processing such as ASB-14-4 to myelin or ethanol or acetone or methanol-ammonium acetate precipitation of myelin proteins. In addition, the step involves no precipitation that selective removal of any proteins as a result of precipitation is nil and a qualitative representation of myelin proteins in a 2-D gel is achieved. PMID:22102008

  20. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S. (Ames, IA); Chang, Huan-Tsang (Silver Spring, MD); Fung, Eliza N. (Ames, IA); Li, Qingbo (Ames, IA); Lu, Xiandan (Ames, IA)

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  1. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Li, Q.; Lu, X.

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  2. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S. (Ames, IA); Li, Qingbo (Ames, IA); Lu, Xiandan (Ames, IA)

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  3. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Chang, H.T.; Fung, E.N.; Li, Q.; Lu, X.

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  4. Investigation of Pokemon-regulated proteins in hepatocellular carcinoma using mass spectrometry-based multiplex quantitative proteomics.

    PubMed

    Bi, Xin; Jin, Yibao; Gao, Xiang; Liu, Feng; Gao, Dan; Jiang, Yuyang; Liu, Hongxia

    2013-01-01

    Pokemon is a transcription regulator involved in embryonic development, cellular differentiation and oncogenesis. It is aberrantly overexpressed in multiple human cancers including Hepatocellular carcinoma (HCC) and is considered as a promising biomarker for HCC. In this work, the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy was used to investigate the proteomic profile associated with Pokemon in human HCC cell line QGY7703 and human hepatocyte line HL7702. Samples were labeled with four-plex iTRAQ reagents followed by two-dimensional liquid chromatography coupled with tandem mass spectrometry analysis. A total of 24 differentially expressed proteins were selected as significant. Nine proteins were potentially up-regulated by Pokemon while 15 proteins were potentially down-regulated and many proteins were previously identified as potential biomarkers for HCC. Gene ontology (GO) term enrichment revealed that the listed proteins were mainly involved in DNA metabolism and biosynthesis process. The changes of glucose-6-phosphate 1-dehydrogenase (G6PD, up-regulated) and ribonucleoside-diphosphate reductase large sub-unit (RIM1, down-regulated) were validated by Western blotting analysis and denoted as Pokemon's function of oncogenesis. We also found that Pokemon potentially repressed the expression of highly clustered proteins (MCM3, MCM5, MCM6, MCM7) which played key roles in promoting DNA replication. Altogether, our results may help better understand the role of Pokemon in HCC and promote the clinical applications. PMID:24261083

  5. Quantitative Changes in In Vitro and In Vivo Protein Synthesis in Aging and Rejuvenated Soybean Cotyledons 1

    PubMed Central

    Skadsen, Ronald W.; Cherry, Joe H.

    1983-01-01

    Cotyledons of light-grown soybean (Glycine max L. var Wayne) seedlings were used as a model system to study the possibility that aging requires qualitative changes in protein synthesis. Cotyledons reached a final stage of senescence and then abscised about 22 days after imbibition. Cotyledon senescence was reversed at 20 days after germination by epicotyl removal. Thereafter, the cotyledons regained much of the chlorophyll, RNA, protein, and polyribosomes lost during aging. Total poly(A)mRNA was extracted from 4-, 12-, 20-day-old, and rejuvenated cotyledons and translated in a wheat germ system. Comparison of translation products on two-dimensional O'Farrell gels showed that many translation products increased in quantity during aging, while roughly half as many decreased. Rejuvenation returned the translation products to approximately 4-day-old levels in roughly half of those products which were diminished with age. Conversely, almost one-third of the products which had increased with age decreased with rejuvenation. None of the translation products were totally lost nor were newly synthesized products detected during aging. Therefore, aging in this system probably does not involve complete gene repression or depression. The observation that epicotyl removal causes a reversal in the levels of various proteins synthesized in vitro was corroborated by similar observations following in vivo labeling of cotyledon sections and analysis by SDS-polyacrylamide gel electrophoresis and fluorography. Densitometric scans of fluorograms revealed a gradual shift in profiles of both in vitro and in vivo translation products during aging. Rejuvenated cotyledon proteins had a profile resembling that of 4-day-old cotyledons. The overall level of [35S]methionine incorporation into protein in vivo declined gradually during aging but was restored to 4-day-old levels within 2 days after epicotyl removal. Images Fig. 3 Fig. 5 Fig. 6 Fig. 7 PMID:16662920

  6. Determination of opium alkaloids in crude opium using non-aqueous capillary electrophoresis

    Microsoft Academic Search

    Inga Bjornsdottir; Steen Honoré Hansen

    1995-01-01

    A method for the quantitative determination of the opium alkaloids morphine, codeine, thebaine, noscapine and papaverine in crude opium and in drug preparations based on non-aqueous capillary electrophoresis has been developed. The non-aqueous mode provides high separation selectivity and new possibilities for regulating the selectivity in capillary electrophoresis. The nature of the organic solvent, the acidity of the electrolytes as

  7. In vivo quantitative proteomics of somatosensory cortical synapses shows which protein levels are modulated by sensory deprivation

    PubMed Central

    Butko, Margaret T.; Savas, Jeffrey N.; Friedman, Beth; Delahunty, Claire; Ebner, Ford; Yates, John R.; Tsien, Roger Y.

    2013-01-01

    Postnatal bilateral whisker trimming was used as a model system to test how synaptic proteomes are altered in barrel cortex by sensory deprivation during synaptogenesis. Using quantitative mass spectrometry, we quantified more than 7,000 synaptic proteins and identified 89 significantly reduced and 161 significantly elevated proteins in sensory-deprived synapses, 22 of which were validated by immunoblotting. More than 95% of quantified proteins, including abundant synaptic proteins such as PSD-95 and gephyrin, exhibited no significant difference under high- and low-activity rearing conditions, suggesting no tissue-wide changes in excitatory or inhibitory synaptic density. In contrast, several proteins that promote mature spine morphology and synaptic strength, such as excitatory glutamate receptors and known accessory factors, were reduced significantly in deprived synapses. Immunohistochemistry revealed that the reduction in SynGAP1, a postsynaptic scaffolding protein, was restricted largely to layer I of barrel cortex in sensory-deprived rats. In addition, protein-degradation machinery such as proteasome subunits, E2 ligases, and E3 ligases, accumulated significantly in deprived synapses, suggesting targeted synaptic protein degradation under sensory deprivation. Importantly, this screen identified synaptic proteins whose levels were affected by sensory deprivation but whose synaptic roles have not yet been characterized in mammalian neurons. These data demonstrate the feasibility of defining synaptic proteomes under different sensory rearing conditions and could be applied to elucidate further molecular mechanisms of sensory development. PMID:23382246

  8. Qualitative and quantitative changes in exoskeletal proteins synthesized throughout the molt cycle of the Bermuda land crab

    SciTech Connect

    Stringfellow, L.A.; Skinner, D.M.

    1987-05-01

    During the premolt period in Crustacea, a single layer of epidermal cells that underlies the exoskeleton is thought to be responsible for the degradation of the old exoskeleton and synthesis of a new one. In order to identify molt-specific proteins and their temporal appearance, they cultured epidermis and associated integumentary tissue from the gill chambers of crab in vitro in the presence of one of three radiolabeled amino acids. Autoradiographs of (/sup 35/S)Met-labeled tissues indicate a low level of synthesis in epidermal cells of intermolt animals; synthesis increases during premolt and stage B of postmolt. Label is also found in the innermost layer of the old exoskeleton while it is being degraded and in new exoskeletal layers during their synthesis. Fluorographs of gels of integumentary proteins show marked quantitative changes in 44 and 56 kD proteins late in premolt. Qualitative changes include synthesis of 46 and 48 kD proteins during late premolt and three proteins (all of approx. 170 kD) detectable only in postmolt. Solubilized gel slices of (/sup 3/H)Leu-labeled proteins indicate maximum synthesis at an earlier premolt stage than seen in Met-labeled proteins. Other proteins of 20, 24, 29, 32, and 96 kD are synthesized in a stage-dependent manner while (/sup 3/H)Tyr labels small proteins that appear only in late premolt.

  9. Two-dimensional gel electrophoresis in proteomics: a tutorial Thierry Rabilloud 1,2

    E-print Network

    Paris-Sud XI, Université de

    1 Two-dimensional gel electrophoresis in proteomics: a tutorial Thierry Rabilloud 1,2 , Cécile)-4-38-78-44-99 e-mail: Thierry.Rabilloud@ cea.fr #12;2 Abstract Two-dimensional electrophoresis of proteins has of the process, from sample preparation to in-gel detection of proteins, commenting the constraints and caveats

  10. Quantitative Co-Expression of Proteins at the Single Cell Level – Application to a Multimeric FRET Sensor

    PubMed Central

    Goedhart, Joachim; van Weeren, Laura; Adjobo-Hermans, Merel J.W.; Elzenaar, Ies; Hink, Mark A.; Gadella, Theodorus W.J.

    2011-01-01

    Background Co-expression of proteins is generally achieved by introducing two (or more) independent plasmids into cells, each driving the expression of a different protein of interest. However, the relative expression levels may vary strongly between individual cells and cannot be controlled. Ideally, co-expression occurs at a defined ratio, which is constant among cells. This feature is of particular importance for quantitative single cell studies, especially those employing bimolecular Förster Resonance Energy Transfer (FRET) sensors. Methodology/Principal Findings Four co-expression strategies based on co-transfection, a dual promotor plasmid, an internal ribosome entry site (IRES) and a viral 2A peptide were selected. Co-expression of two spectrally separable fluorescent proteins in single living cells was quantified. It is demonstrated that the 2A peptide strategy can be used for robust equimolar co-expression, while the IRES sequence allows expression of two proteins at a ratio of approximately 3:1. Combined 2A and IRES elements were used for the construction of a single plasmid that drives expression of three individual proteins, which generates a FRET sensor for measuring heterotrimeric G-protein activation. The plasmid drives co-expression of donor and acceptor tagged subunits, with reduced heterogeneity, and can be used to measure G-protein activation in single living cells. Conclusions/Significance Quantitative co-expression of two or more proteins can be achieved with little cell-to-cell variability. This finding enables reliable co-expression of donor and acceptor tagged proteins for FRET studies, which is of particular importance for the development of novel bimolecular sensors that can be expressed from single plasmid. PMID:22114669

  11. Using capillary electrophoresis for failure analysis

    SciTech Connect

    Kelly, R.G.; Scully, H.S.; Stoner, G.E. (Univ. of Virginia, Charlottesville, VA (United States). Center for Electrochemical Science and Engineering)

    1993-07-01

    Capillary electrophoresis (CE), an advanced solution analysis technique, can be used for failure analysis of corroded components. It has high sensitivity (concentrations as low as parts-per-trillion) and can detect quantitatively a large number of ionic species. CE determined the vapor-phase attack by organic acids, mainly acetic acid, on an electrical equipment enclosure. These acids most likely originated from the seasoning of the oak pallets used to transport the manufactured items, accumulating inside the shrink-wrap film used to bind packages to the pallet.

  12. Quantitative measurements of the cooperativity in an EF-hand protein with sequential calcium binding.

    PubMed Central

    Linse, S.; Chazin, W. J.

    1995-01-01

    Positive cooperativity, defined as an enhancement of the ligand affinity at one site as a consequence of binding the same type of ligand at another site, is a free energy coupling between binding sites. It can be present both in systems with sites having identical ligand affinities and in systems where the binding sites have different affinities. When the sites have widely different affinities such that they are filled with ligand in a sequential manner, it is often difficult to quantify or even detect the positive cooperativity, if it occurs. This study presents verification and quantitative measurements of the free energy coupling between the two calcium binding sites in a mutant form of calbindin D9k. Wild-type calbindin D9k binds two calcium ions with similar affinities and positive cooperativity--the free energy coupling, delta delta G, is around -8 kJ.mol-1 (Linse S, et al., 1991, Biochemistry 30: 154-162). The mutant, with the substitution Asn 56-->Ala, binds calcium in a sequential manner. In the present work we have taken advantage of the variations among different metal ions in terms of their preferences for the two binding sites in calbindin D9k. Combined studies of the binding of Ca2+, Cd2+, and La3+ have allowed us to conclude that in this mutant delta delta G < -6.4 kJ.mol-1, and that Cd2+ and La3+ also bind to this protein with positive cooperativity. The results justify the use of the (Ca2+)1 state of the Asn 56-->Ala mutant, as well as the (Cd2+)1 state of the wild type, as models for the half-saturated states along the two pathways of cooperative Ca2+ binding in calbindin D9k. PMID:7549868

  13. Improved Methodical Approach for Quantitative BRET Analysis of G Protein Coupled Receptor Dimerization

    PubMed Central

    Szalai, Bence; Hoffmann, Péter; Prokop, Susanne; Erdélyi, László; Várnai, Péter; Hunyady, László

    2014-01-01

    G Protein Coupled Receptors (GPCR) can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V2 vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, ?2 adrenergic and CB1 cannabinoid receptors. PMID:25329164

  14. Comparing SILAC and two-dimensional gel electrophoresis image analysis for profiling urokinase plasminogen activator signaling in ovarian cancer cells.

    PubMed

    Uitto, Pauliina M; Lance, Braddon K; Wood, Graham R; Sherman, James; Baker, Mark S; Molloy, Mark P

    2007-06-01

    Two-dimensional gel electrophoresis (2-DE) image analysis is conventionally used for comparative proteomics. However, there are a number of technical difficulties associated with 2-DE protein separation that limit the depth of proteome coverage, and the image analysis steps are typically labor-intensive and low-throughput. Recently, mass spectrometry-based quantitation strategies have been described as alternative differential proteome analysis techniques. In this study, we investigated changes in protein expression using an ovarian cancer cell line, OVMZ6, 24 h post-stimulation with the relatively weak agonist, urokinase-type plasminogen activator (uPA). Quantitative protein profiles were obtained by MALDI-TOF/TOF from stable isotope-labeled cells in culture (SILAC), and these results were compared to the quantitative ratios obtained using 2-DE gel image analysis. MALDI-TOF/TOF mass spectrometry showed that differential quantitation using SILAC was highly reproducible (approximately 8% coefficient of variation (CV)), and this variance was considerably lower than that achieved using automated 2-DE image analysis strategies (CV approximately 25%). Both techniques revealed subtle alterations in cellular protein expression following uPA stimulation. However, due to the lower variances associated with the SILAC technique, smaller changes in expression of uPA-inducible proteins could be found with greater certainty. PMID:17472359

  15. Alignment of two-dimensional electrophoresis gels Guihua Shi, Tianzi Jiang *, Wanlin Zhu, Bing Liu, Huizhi Zhao

    E-print Network

    Jiang,Tianzi

    Alignment of two-dimensional electrophoresis gels Guihua Shi, Tianzi Jiang *, Wanlin Zhu, Bing Liu-dimensional electrophoresis is a major separating technique for proteins in proteomics. Alignment of gel images is critical a novel iterative closest point (ICP) method for 2D-gel electrophoresis image alignment. The paper seeks

  16. Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

    PubMed

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

    2015-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification. PMID:25223624

  17. Differential Protein Expression Profiles in Estrogen Receptor–Positive and –Negative Breast Cancer Tissues Using Label-Free Quantitative Proteomics

    PubMed Central

    Rezaul, Karim; Thumar, Jay Kumar; Lundgren, Deborah H.; Eng, Jimmy K.; Claffey, Kevin P.; Wilson, Lori; Han, David K.

    2010-01-01

    Identification of the proteins that are associated with estrogen receptor (ER) status is a first step towards better understanding of the hormone-dependent nature of breast carcinogenesis. Although a number of gene expression analyses have been conducted, protein complement has not been systematically investigated to date. Because proteins are primary targets of therapeutic drugs, in this study, we have attempted to identify proteomic signatures that demarcate ER-positive and -negative breast cancers. Using highly enriched breast tumor cells, replicate analyses from 3 ER?+ and 3 ER?? human breast tumors resulted in the identification of 2,995 unique proteins with ?2 peptides. Among these, a number of receptor tyrosine kinases and intracellular kinases that are abundantly expressed in ER?+ and ER?? breast cancer tissues were identified. Further, label-free quantitative proteome analysis revealed that 236 proteins were differentially expressed in ER?+ and ER?? breast tumors. Among these, 141 proteins were selectively up-regulated in ER?+, and 95 proteins were selectively up-regulated in ER?? breast tumors. Comparison of differentially expressed proteins with a breast cancer database revealed 98 among these have been previously reported to be involved in breast cancer. By Gene Ontology molecular function, dehydrogenase, reductase, cytoskeletal proteins, extracellular matrix, hydrolase, and lyase categories were significantly enriched in ER?+, whereas selected calcium-binding protein, membrane traffic protein, and cytoskeletal protein were enriched in ER?? breast tumors. Biological process and pathway analysis revealed that up-regulated proteins of ER?+ were overrepresented by proteins involved in amino acid metabolism, proteasome, and fatty acid metabolism, while up-regulated proteins of ER?? were overrepresented by proteins involved in glycolysis pathway. The presence and relative abundance of 4 selected differentially abundant proteins (liprin-?1, fascin, DAP5, and ?-arrestin-1) were quantified and validated by immunohistochemistry. In conclusion, unlike in vitro cell culture models, the in vivo signaling proteins and pathways that we have identified directly from human breast cancer tissues may serve as relevant therapeutic targets for the pharmacological intervention of breast cancer. PMID:21779449

  18. Quantitative Profiling of In Vivo-assembled RNA-Protein Complexes Using a Novel Integrated Proteomic Approach*

    PubMed Central

    Tsai, Becky Pinjou; Wang, Xiaorong; Huang, Lan; Waterman, Marian L.

    2011-01-01

    Identification of proteins in RNA-protein complexes is an important step toward understanding regulation of RNA-based processes. Because of the lack of appropriate methodologies, many studies have relied on the creation of in vitro assembled RNA-protein complexes using synthetic RNA and cell extracts. Such complexes may not represent authentic RNPs as they exist in living cells as synthetic RNA may not fold properly and nonspecific RNA-protein interactions can form during cell lysis and purification processes. To circumvent limitations in current approaches, we have developed a novel integrated strategy namely MS2 in vivo biotin tagged RNA affinity purification (MS2-BioTRAP) to capture bona fide in vivo-assembled RNA-protein complexes. In this method, HB-tagged bacteriophage protein MS2 and stem-loop tagged target or control RNAs are co-expressed in cells. The tight association between MS2 and the RNA stem-loop tags allows efficient HB-tag based affinity purification of authentic RNA-protein complexes. Proteins associated with target RNAs are subsequently identified and quantified using SILAC-based quantitative mass spectrometry. Here the 1.2 kb internal ribosome entry site (IRES) from lymphoid enhancer factor-1 mRNA has been used as a proof-of-principle target RNA. An IRES target was chosen because of its importance in protein translation and our limited knowledge of proteins associated with IRES function. With a conventionally translated target RNA as control, 36 IRES binding proteins have been quantitatively identified including known IRES binding factors, novel interacting proteins, translation initiation factors (eIF4A-1, eIF-2A, and eIF3g), and ribosomal subunits with known noncanonical actions (RPS19, RPS7, and RPL26). Validation studies with the small molecule eIF4A-1 inhibitor Hippuristanol shows that translation of endogenous lymphoid enhancer factor-1 mRNA is especially sensitive to eIF4A-1 activity. Our work demonstrates that MS2 in vivo biotin tagged RNA affinity purification is an effective and versatile approach that is generally applicable for other RNA-protein complexes. PMID:21285413

  19. Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes.

    PubMed

    Buenrostro, Jason D; Araya, Carlos L; Chircus, Lauren M; Layton, Curtis J; Chang, Howard Y; Snyder, Michael P; Greenleaf, William J

    2014-06-01

    RNA-protein interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. Here we repurpose a high-throughput sequencing instrument to quantitatively measure binding and dissociation of a fluorescently labeled protein to >10(7) RNA targets generated on a flow cell surface by in situ transcription and intermolecular tethering of RNA to DNA. Studying the MS2 coat protein, we decompose the binding energy contributions from primary and secondary RNA structure, and observe that differences in affinity are often driven by sequence-specific changes in both association and dissociation rates. By analyzing the biophysical constraints and modeling mutational paths describing the molecular evolution of MS2 from low- to high-affinity hairpins, we quantify widespread molecular epistasis and a long-hypothesized, structure-dependent preference for G:U base pairs over C:A intermediates in evolutionary trajectories. Our results suggest that quantitative analysis of RNA on a massively parallel array (RNA-MaP) provides generalizable insight into the biophysical basis and evolutionary consequences of sequence-function relationships. PMID:24727714

  20. Depletion theory of protein transport in semi-dilute polymer solutions.

    PubMed Central

    Odijk, T

    2000-01-01

    We consider the effect of polymer depletion on the transport (diffusion and electrophoresis) of small proteins through semi-dilute solutions of a flexible polymer. A self-consistent field theory may be set up in the important case of quasi-ideal interactions when the protein is small enough. Dynamic depletion, the reorganization of the depletion layer as the protein diffuses, is computed within a free-draining approximation. The transport of the dressed particle (protein + depletion layer) is tackled by extending Ogston's analysis of probe diffusion through fibrous networks to the case of a probe diffusing through a semi-dilute polymer inhomogeneous on the scale of the polymer correlation length. The resulting exponential retardation agrees almost quantitatively with that found in recent electrophoresis experiments of small proteins in polymer solutions that have been ascertained to be semi-dilute (S. P. Radko and A. Chrambach, Electrophoresis, 17:1094-1102, 1996; Biopolymers, 4:183-189, 1997). PMID:11053111

  1. Static continuous electrophoresis device

    NASA Technical Reports Server (NTRS)

    Rhodes, P. H. (inventor)

    1982-01-01

    An apparatus is disclosed for carrying out a moving wall type electrophoresis process for separation of cellular particles. The apparatus includes a water-tight housing containing an electrolytic buffer solution. A separation chamber in the housing is defined by spaced opposed moving walls and spaced opposed side walls. Substrate assemblies, which support the moving wall include vacuum ports for positively sealing the moving walls against the substrate walls. Several suction conduits communicate with the suction ports and are arranged in the form of valleys in a grid plate. The raised land portion of the grid plat supports the substrate walls against deformation inwardly under suction. A cooling chamber is carried on the back side of plate. The apparatus also has tensioner means including roller and adjustment screws for maintaining the belts in position and a drive arrangement including an electric motor with a gear affixed to its output shaft. Electrode assemblies are disposed to provide the required electric field.

  2. Electrophoresis experiment for space

    NASA Technical Reports Server (NTRS)

    Vanderhoff, J. W.; Micale, F. J.

    1976-01-01

    The Apollo 16 electrophoresis experiment was analyzed, demonstrating that the separation of the two different-size monodisperse latexes did indeed take place, but that the separation was obscured by the pronounced electroosmotic flow of the liquid medium. The results of this experiment, however, were dramatic since it is impossible to carry out a similar separation on earth. It can be stated unequivocally from this experiment that any electrophoretic separation will be enhanced under microgravity conditions. The only question is the degree of this enhancement, which can be expected to vary from one experimental technique to another. The low-electroosmotic-mobility coating (Z6040-MC) developed under this program was found to be suitable for a free-fluid electrophoretic separation such as the experiment designed for the ASTP flight. The problem with this coating, however, is that its permanency is limited because of the slow desorption of the methylcellulose from the coated surface.

  3. Electrophoresis demonstration on Apollo 16

    NASA Technical Reports Server (NTRS)

    Snyder, R. S.

    1972-01-01

    Free fluid electrophoresis, a process used to separate particulate species according to surface charge, size, or shape was suggested as a promising technique to utilize the near zero gravity condition of space. Fluid electrophoresis on earth is disturbed by gravity-induced thermal convection and sedimentation. An apparatus was developed to demonstrate the principle and possible problems of electrophoresis on Apollo 14 and the separation boundary between red and blue dye was photographed in space. The basic operating elements of the Apollo 14 unit were used for a second flight demonstration on Apollo 16. Polystyrene latex particles of two different sizes were used to simulate the electrophoresis of large biological particles. The particle bands in space were extremely stable compared to ground operation because convection in the fluid was negligible. Electrophoresis of the polystyrene latex particle groups according to size was accomplished although electro-osmosis in the flight apparatus prevented the clear separation of two particle bands.

  4. Kidney cell electrophoresis, continuing task

    NASA Technical Reports Server (NTRS)

    Todd, P. W.

    1985-01-01

    Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated to provide ground support in the form of analytical cell electrophoresis and flow cytometry. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. Cells were prepared in suspension prior to flight in electrophoresis buffer and 10% calf serum. Electrophoretic separation proceeded in electrophoresis buffer without serum in the Continuous Flow Electrophoretic Separator, and fractions were collected into sample bags containing culture medium and concentrated serum. Fractions that yielded enough progeny cells were analyzed for morphology and electrophoretic mobility distributions. It is noted that the lowest mobility fraction studied produced higher mobility progeny while the other fractions produced progeny cells with mobilities related to the fractions from which they were collected.

  5. Quantitation of free polyethylene glycol in PEGylated protein conjugate by size exclusion HPLC with refractive index (RI) detection.

    PubMed

    Li, Ning; Ziegemeier, Daisy; Bass, Laura; Wang, Wei

    2008-12-15

    In this study, size exclusion high performance liquid chromatography was evaluated for its application in separation and quantitation of free polyethylene glycol (PEG) and its PEGylated-protein-conjugate (PEG-conjugate). Although the large mass of the free PEG (2-fold greater than the protein) made separation difficult, chromatographic conditions were identified enabling resolution and quantitation of the free PEG, PEG-conjugate and non-PEGylated protein with Shodex Protein KW803 and KW804 columns in series and refractive index detection. The optimum resolution of 1.7 and 2.0 was achieved for the free PEG and PEG-conjugate as well as the free PEG and non-PEGylated protein using 20mM HEPES buffer at pH 6.5. Under this condition, the plot of log(10)MW of all the pertinent analytes against retention time showed a linear relationship with a correlation coefficient of 1. Limited assay performance evaluation demonstrated that the method was linear in the concentration range of 10 to 250 microg/mL of free PEG with correlation coefficients of > or = 0.99. When free PEG in this concentration range was spiked into PEG-conjugate samples at 1mg/mL, the recovery was in the range of 78%-120%. Detection and quantitation limits were determined to be, respectively, 10 and 25 microg/mL for free PEG. The R.S.D. for intra- and inter-day precision was 0.09% or less for retention time measurements and 2.9% or less for area count measurements. Robustness testing was performed by deliberately deviating +/-0.2 pH units away from the desired pH as well as by increasing the flow rate. These deviations resulted in no significant impact on area percent distribution of all species. However, separation was found to be sensitive to high ionic strength and buffer species. PMID:19019609

  6. Expanding the capabilities of microfluidic gradient elution moving boundary electrophoresis for complex samples.

    PubMed

    Strychalski, Elizabeth A; Henry, Alyssa C; Ross, David

    2011-08-15

    Gradient elution moving boundary electrophoresis (GEMBE) is a robust, continuous injection separation technique that uses electrophoresis to drive electrically charged analytes into a capillary or microfluidic channel for detection, while opposing electroosmosis and controlled variable pressure-driven flow prevent other sample components-for example, cells, proteins, or particulates in complex samples that can interfere with analysis-from entering the channel. This work expands the sample-in/answer-out analytical capabilities of GEMBE for complex samples by demonstrating the quantitative analysis of anions, implementing aqueous background electrolyte (BGE) solutions at neutral pH, and introducing the use of additives to the sample solution to optimize performance. Dirt was analyzed quantitatively, with the sole preparatory step of suspension in an aqueous BGE solution at neutral pH, for dissolved chloride, nitrite, nitrate, sulfate, and oxalate using GEMBE with capacitively-coupled contactless conductivity detection. In addition to altering the pH of the BGE solution, optimization of the analysis of dirt and whole blood was achieved using various commercially available additives. These results, taken together with previous demonstrations of GEMBE for the analysis of complex samples, underscore the uncomplicated versatility of GEMBE, facilitate effective analysis of biological complex samples using BGE solutions at physiological pH, and offer a sufficient set of techniques and tools to build a foundation for the analysis of a broad range of complex samples. PMID:21766783

  7. Colostrum protein uptake in neonatal lambs examined by descriptive and quantitative liquid chromatography-tandem mass spectrometry.

    PubMed

    Hernández-Castellano, Lorenzo E; Argüello, Anastasio; Almeida, André M; Castro, Noemí; Bendixen, Emøke

    2015-01-01

    Colostrum intake is a key factor for newborn ruminant survival because the placenta does not allow the transfer of immune components. Therefore, newborn ruminants depend entirely on passive immunity transfer from the mother to the neonate, through the suckling of colostrum. Understanding the importance of specific colostrum proteins has gained significant attention in recent years. However, proteomics studies of sheep colostrum and their uptake in neonate lambs has not yet been presented. The aim of this study was to describe the proteomes of sheep colostrum and lamb blood plasma, using sodium dodecyl sulfate-PAGE for protein separation and in-gel digestion, followed by liquid chromatography-tandem mass spectrometry of resulting tryptic peptides for protein identification. An isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics approach was subsequently used to provide relative quantification of how neonatal plasma protein concentrations change as an effect of colostrum intake. The results of this study describe the presence of 70 proteins in the ovine colostrum proteome. Furthermore, colostrum intake resulted in an increase of 8 proteins with important immune functions in the blood plasma of lambs. Further proteomic studies will be necessary, particularly using the selected reaction monitoring approach, to describe in detail the role of specific colostrum proteins for immune transfer to the neonate. PMID:25465637

  8. WebProAnalyst: an interactive tool for analysis of quantitative structure–activity relationships in protein families

    PubMed Central

    Ivanisenko, Vladimir A.; Eroshkin, Alexey M.; Kolchanov, Nickolay A.

    2005-01-01

    WebProAnalyst is a web-accessible analysis tool () designed for scanning quantitative structure–activity relationships in protein families. The tool allows users to search correlations between protein activity and physicochemical characteristics (i.e. hydrophobicity or alpha-helical amphipathicity) in queried sequences. WebProAnalyst uses aligned amino acid sequences and data on protein activity (pK, Km, ED50, among others). WebProAnalyst implements methods of the known ProAnalyst package, including the multiple linear regression analysis and the sequence–activity correlation coefficient. In addition, WebProAnalyst incorporates a method based on neural networks. The WebProAnalyst reports a list of sites in protein family, the regression analysis parameters (including correlation values) for the relationships between the amino acid physicochemical characteristics in the site and the protein activity values. WebProAnalyst is useful in search of the amino acid residues that are important for protein function/activity. Furthermore, WebProAnalyst may be helpful in designing the protein-engineering experiments. PMID:15980590

  9. Evaluation of the Taxonomic Relationship of Micrococcus cryoph ilus, Branhamella catarrhalis, and Neisseriae by Comparative Polyacrylamide Gel Electrophoresis of Soluble Proteins

    Microsoft Academic Search

    RICHARD H. FOX

    Electrophoretic profiles of soluble proteins derived from seven strains of Bran ham el la ca tarr halis, Ne isseria perflava, Neisseria sicca, an d the psy c hr o p hile , Micrococcus cry ophilus, were compared. The profiles produced from the strains of Branhamella catarrhalis showed a marked similarity except for that of strain Ne4 (ATCC 23246) which was

  10. Quantitative mass spectrometry and PAR-CLIP to identify RNA-protein interactions.

    PubMed

    Scheibe, Marion; Butter, Falk; Hafner, Markus; Tuschl, Thomas; Mann, Matthias

    2012-10-01

    Systematic analysis of the RNA-protein interactome requires robust and scalable methods. We here show the combination of two completely orthogonal, generic techniques to identify RNA-protein interactions: PAR-CLIP reveals a collection of RNAs bound to a protein whereas SILAC-based RNA pull-downs identify a group of proteins bound to an RNA. We investigated binding sites for five different proteins (IGF2BP1-3, QKI and PUM2) exhibiting different binding patterns. We report near perfect agreement between the two approaches. Nevertheless, they are non-redundant, and ideally complement each other to map the RNA-protein interaction network. PMID:22885304

  11. ProRata: A quantitative proteomics program for accurate protein abundance ratio estimation with confidence interval evaluation

    SciTech Connect

    Pan, Chongle [ORNL; Kora, Guruprasad H [ORNL; McDonald, W Hayes [ORNL; Tabb, Dave L [ORNL; Verberkmoes, Nathan C [ORNL; Hurst, Gregory {Greg} B [ORNL; Pelletier, Dale A [ORNL; Samatova, Nagiza F [ORNL; Hettich, Robert {Bob} L [ORNL

    2006-01-01

    A profile likelihood algorithm is proposed for quantitative shotgun proteomics to infer the abundance ratios of proteins from the abundance ratios of isotopically labeled peptides derived from proteolysis. Previously, we have shown that the estimation variability and bias of peptide abundance ratios can be predicted from their profile signal-to-noise ratios. Given multiple quantified peptides for a protein, the profile likelihood algorithm probabilistically weighs the peptide abundance ratios by their inferred estimation variability, accounts for their expected estimation bias, and suppresses contribution from outliers. This algorithm yields maximum likelihood point estimation and profile likelihood confidence interval estimation of protein abundance ratios. This point estimator is more accurate than an estimator based on the average of peptide abundance ratios. The confidence interval estimation provides an "error bar" for each protein abundance ratio that reflects its estimation precision and statistical uncertainty. The accuracy of the point estimation and the precision and confidence level of the interval estimation were benchmarked with standard mixtures of isotopically labeled proteomes. The profile likelihood algorithm was integrated into a quantitative proteomics program, called ProRata, freely available at www.MSProRata.org.

  12. Measurement of local rates of brain protein synthesis by quantitative autoradiography: validation with L-(/sup 3/H)valine

    SciTech Connect

    Dwyer, B.E.; Donatoni, P.; Wasterlain, C.G.

    1982-12-01

    Following the injection of 4-day old rats with 150 mM L-(3,4-/sup 3/H)valine (10 mumol/g, IP) the incorporation of /sup 3/H into protein was linear 2 hours. Valine specific activity in the brain acid-soluble fraction was constant between 30 and 120 min after injection with a mean value of 82.3% of the injectate. Significant amounts of tritated metabolites accumulated in the brain acid-soluble fraction (41.4% of radioactivity at 120 min) but do not prove an impediment to measuring rates of protein synthesis. The rate of protein synthesis in cerebral cortex of the 4-day old rat was measured by quantitative autoradiography using (/sup 3/H)valine and /sup 3/H-sensitive film. The measured rate shows excellent agreement with that found previously using L-(1-/sup 14/C)valine. Our results suggest that (/sup 3/H)valine can be a useful precursor to measure local rates of brain protein synthesis by quantitative autoradiography.

  13. Quantitative proteomic analysis of primary neurons reveals diverse changes in synaptic protein content in fmr1 knockout mice

    PubMed Central

    Liao, Lujian; Park, Sung Kyu; Xu, Tao; Vanderklish, Peter; Yates, John R.

    2008-01-01

    Fragile X syndrome (FXS) is a common inherited form of mental retardation that is caused, in the vast majority of cases, by the transcriptional silencing of a single gene, fmr1. The encoded protein, FMRP, regulates mRNA translation in neuronal dendrites, and it is thought that changes in translation-dependent forms of synaptic plasticity lead to many symptoms of FXS. However, little is known about the potentially extensive changes in synaptic protein content that accompany loss of FMRP. Here, we describe the development of a high-throughput quantitative proteomic method to identify differences in synaptic protein expression between wild-type and fmr1?/? mouse cortical neurons. The method is based on stable isotope labeling by amino acids in cell culture (SILAC), which has been used to characterize differentially expressed proteins in dividing cells, but not in terminally differentiated cells because of reduced labeling efficiency. To address the issue of incomplete labeling, we developed a mathematical method to normalize protein ratios relative to a reference based on the labeling efficiency. Using this approach, in conjunction with multidimensional protein identification technology (MudPIT), we identified >100 proteins that are up- or down-regulated. These proteins fall into a variety of functional categories, including those regulating synaptic structure, neurotransmission, dendritic mRNA transport, and several proteins implicated in epilepsy and autism, two endophenotypes of FXS. These studies provide insights into the potential origins of synaptic abnormalities in FXS and a demonstration of a methodology that can be used to explore neuronal protein changes in neurological disorders. PMID:18829439

  14. Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR

    E-print Network

    Zhou, Kang

    Background: Accurate interpretation of quantitative PCR (qPCR) data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. ...

  15. A Quantitative Description of the Binding States and In Vitro Function of Antitermination Protein N of Bacteriophage ?

    PubMed Central

    Conant, Clarke R.; Van Gilst, Marc R.; Weitzel, Stephen E.; Rees, William A.; von Hippel, Peter H.

    2008-01-01

    The N protein of bacteriophage ? activates transcription of genes that lie downstream of termination sequences by suppressing transcription termination. N binds to specific (boxB) and non-specific sites on the transcript RNA and contacts RNA polymerase via cis-RNA looping, resulting in “antitermination” of transcription. To find the effect of N–boxB binding on antitermination, we quantitatively relate binding measurements made in isolation to in vitro antitermination activity. We measure binding of N to boxB RNA, non-specific single-stranded RNA, and non-specific double-stranded DNA fluorimetrically, and use an equilibrium model to describe quantitatively the binding of N to nucleic acids of Escherichia coli transcription elongation complexes. We then test the model by comparison with in vitro N antitermination activity measured in reactions containing these same elongation complexes. We find that binding of N protein to the nucleic acid components of transcription elongation complexes can quantitatively predict antitermination activity, suggesting that antitermination in vitro is determined by a nucleic acid binding equilibrium with one molecule of N protein per RNA transcript being sufficient for antitermination. Elongation complexes contain numerous overlapping non-specific RNA and DNA-binding sites for N; the large number of sites compensates for the low N binding affinity, so multiple N proteins are expected to bind to elongation complexes. The occupancy/activity of these proteins is described by a binomial distribution of proteins on transcripts containing multiple non-specific sites. The contribution of specific (boxB) binding to activity also depends on this distribution. Specificity is not measured accurately by measurements made in the presence and in the absence of boxB. We find that antitermination is inhibited by non-productive binding of N to non-specific sites on template DNA, and that NusA protein covers RNA sites on the transcript, limiting N access and activity. The activity and specificity of regulatory proteins that loop from high-affinity binding sites are likely modulated by multiple non-specific binding events; in vivo activity may also be regulated by the modulation of non-specific binding. PMID:15854643

  16. Profiling of experience-regulated proteins in the songbird auditory forebrain using quantitative proteomics

    E-print Network

    Jarvis, Erich D.

    -dimensional differential in-gel expression (2D-DIGE) experiments, extensive protein quantification analyses, and tandem processing of vocal communication signals in freely behaving songbirds triggers a cascade of protein

  17. Quantitative label free high throughput protein arrays M. S. nl1,3,2

    E-print Network

    diagnosis. Immunoassays that identify the presence of specific protein biomarkers in blood, urine, or saliva constitute a vital tool in modern healthcare. Solid phase immunoassays for diagnosis. Current Immunoassays generally identify one protein target per test. Clinicians

  18. Protein analysis by 31p NMR spectroscopy in ionic liquid: quantitative determination of enzymatically created cross-links.

    PubMed

    Monogioudi, Evanthia; Permi, Perttu; Filpponen, Ilari; Lienemann, Michael; Li, Bin; Argyropoulos, Dimitris; Buchert, Johanna; Mattinen, Maija-Liisa

    2011-02-23

    Cross-linking of ?-casein by Trichoderma reesei tyrosinase (TrTyr) and Streptoverticillium mobaraense transglutaminase (Tgase) was analyzed by (31)P nuclear magnetic resonance (NMR) spectroscopy in ionic liquid (IL). According to (31)P NMR, 91% of the tyrosine side chains were cross-linked by TrTyr at high dosages. When Tgase was used, no changes were observed because a different cross-linking mechanism was operational. However, this verified the success of the phosphitylation of phenolics within the protein matrix in the IL. Atomic force microscopy (AFM) in solid state showed that disk-shaped nanoparticles were formed in the reactions with average diameters of 80 and 20 nm for TrTyr and Tgase, respectively. These data further advance the current understanding of the action of tyrosinases on proteins on molecular and chemical bond levels. Quantitative (31)P NMR in IL was shown to be a simple and efficient method for the study of protein modification. PMID:21218836

  19. Application and challenges in using LC-MS assays for absolute quantitative analysis of therapeutic proteins in drug discovery.

    PubMed

    Zheng, Joanna; Mehl, John; Zhu, Yongxin; Xin, Baomin; Olah, Timothy

    2014-03-01

    As more protein therapeutics enter the drug-discovery pipeline, the traditional ligand-binding assay (LBA) faces additional challenges to meet the rapid and diverse bioanalytical needs in the early drug-discovery stage. The high specificity and sensitivity afforded by LC-MS, along with its rapid method development, is proving invaluable for the analysis of protein therapeutics in support of drug discovery. LC-MS not only serves as a quantitative tool to complement LBA in drug discovery, it also provides structural details at a molecular level, which are used to address issues that cannot be resolved using LBA alone. This review will describe the key benefits and applications, as well as the techniques and challenges for applying LC-MS to support protein quantification in drug discovery. PMID:24702115

  20. The development and application of a quantitative peptide microarray based approach to protein interaction domain specificity space.

    PubMed

    Engelmann, Brett W; Kim, Yohan; Wang, Miaoyan; Peters, Bjoern; Rock, Ronald S; Nash, Piers D

    2014-12-01

    Protein interaction domain (PID) linear peptide motif interactions direct diverse cellular processes in a specific and coordinated fashion. PID specificity, or the interaction selectivity derived from affinity preferences between possible PID-peptide pairs is the basis of this ability. Here, we develop an integrated experimental and computational cellulose peptide conjugate microarray (CPCMA) based approach for the high throughput analysis of PID specificity that provides unprecedented quantitative resolution and reproducibility. As a test system, we quantify the specificity preferences of four Src Homology 2 domains and 124 physiological phosphopeptides to produce a novel quantitative interactome. The quantitative data set covers a broad affinity range, is highly precise, and agrees well with orthogonal biophysical validation, in vivo interactions, and peptide library trained algorithm predictions. In contrast to preceding approaches, the CPCMAs proved capable of confidently assigning interactions into affinity categories, resolving the subtle affinity contributions of residue correlations, and yielded predictive peptide motif affinity matrices. Unique CPCMA enabled modes of systems level analysis reveal a physiological interactome with expected node degree value decreasing as a function of affinity, resulting in minimal high affinity binding overlap between domains; uncover that Src Homology 2 domains bind ligands with a similar average affinity yet strikingly different levels of promiscuity and binding dynamic range; and parse with unprecedented quantitative resolution contextual factors directing specificity. The CPCMA platform promises broad application within the fields of PID specificity, synthetic biology, specificity focused drug design, and network biology. PMID:25135669

  1. A Critical Appraisal of Quantitative Studies of Protein Degradation in the Framework of Cellular Proteostasis

    PubMed Central

    Alvarez-Castelao, Beatriz; Ruiz-Rivas, Carmen; Castaño, José G.

    2012-01-01

    Protein homeostasis, proteostasis, is essential to understand cell function. Protein degradation is a crucial component of the proteostatic mechanisms of the cell. Experiments on protein degradation are nowadays present in many investigations in the field of molecular and cell biology. In the present paper, we focus on the different experimental approaches to study protein degradation and present a critical appraisal of the results derived from steady-state and kinetic experiments using detection of unlabelled and labelled protein methodologies with a proteostatic perspective. This perspective allows pinpointing the limitations in interpretation of results and the need of further experiments and/or controls to establish “definitive evidence” for the role of protein degradation in the proteostasis of a given protein or the entire proteome. We also provide a spreadsheet for simple calculations of mRNA and protein decays for mimicking different experimental conditions and a checklist for the analysis of experiments dealing with protein degradation studies that may be useful for researchers interested in the area of protein turnover. PMID:23119163

  2. Approach to quantitative detection of CD146 with the label-free protein biosensor based on imaging ellipsometry

    NASA Astrophysics Data System (ADS)

    Niu, Yu; Liu, Li; Yan, Xiyun; Jin, Gang

    2011-03-01

    CD146 glycoprotein belonging to cell adhesion molecules is considered to be a novel target on endothelial cell involved in tumor angiogenesis. The biosensor based on imaging ellipsometry (BIE) which is performed in null and off-null mode is used for CD146 detection as a trial by the following steps. Firstly, anti-CD146 antibody as ligand is immobilized on Protein G modified silicon substrate. Then, CD146 test is carried out and its calibration curve is established for the requirement of quantitative detection. Finally, 18 serum samples are detected quantitatively and their results are validated by ELISA's. The sensitivity for CD146 detection achieves the order of ng/ml and the relationship between BIE signal y (grayscale value) and CD146 concentration x (ng/ml) is y=3.3ln(x) +91.3. Compared with ELISA's, the majority of results are in agreement, and the results of two approaches have significant statistic relevance.

  3. Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells.

    PubMed

    Yang, Jing; Tallman, Keri A; Porter, Ned A; Liebler, Daniel C

    2015-03-01

    Protein alkylation by 4-hydroxy-2-nonenal (HNE), an endogenous lipid derived electrophile, contributes to stress signaling and cellular toxicity. Although previous work has identified protein targets for HNE alkylation, the sequence specificity of alkylation and dynamics in a cellular context remain largely unexplored. We developed a new quantitative chemoproteomic platform, which uses isotopically tagged, photocleavable azido-biotin reagents to selectively capture and quantify the cellular targets labeled by the alkynyl analogue of HNE (aHNE). Our analyses site-specifically identified and quantified 398 aHNE protein alkylation events (386 cysteine sites and 12 histidine sites) in intact cells. This data set expands by at least an order of magnitude the number of such modification sites previously reported. Although adducts formed by Michael addition are thought to be largely irreversible, we found that most aHNE modifications are lost rapidly in situ. Moreover, aHNE adduct turnover occurs only in intact cells and loss rates are site-selective. This quantitative chemoproteomics platform provides a versatile general approach to map bioorthogonal-chemically engineered post-translational modifications and their cellular dynamics in a site-specific and unbiased manner. PMID:25654326

  4. Assessment of ERCC1 and XPF Protein Expression Using Quantitative Immunohistochemistry in Nasopharyngeal Carcinoma Patients Undergoing Curative Intent Treatment

    SciTech Connect

    Jagdis, Amanda [Department of Internal Medicine, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia (Canada)] [Department of Internal Medicine, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia (Canada); Phan, Tien [Department of Radiation Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada) [Department of Radiation Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Faculty of Medicine, University of Calgary, Calgary, Alberta (Canada); Klimowicz, Alexander C. [Department of Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada) [Department of Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Faculty of Medicine, University of Calgary, Calgary, Alberta (Canada); Laskin, Janessa J. [Department of Medical Oncology, British Columbia Cancer Agency–Vancouver, Vancouver, British Columbia (Canada) [Department of Medical Oncology, British Columbia Cancer Agency–Vancouver, Vancouver, British Columbia (Canada); Faculty of Medicine, University of British Columbia, Vancouver, British Columbia (Canada); Lau, Harold Y. [Department of Radiation Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada) [Department of Radiation Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Faculty of Medicine, University of Calgary, Calgary, Alberta (Canada); Petrillo, Stephanie K. [Functional Tissue Imaging Unit, Translational Research Laboratory, Tom Baker Cancer Centre, Calgary, Alberta (Canada)] [Functional Tissue Imaging Unit, Translational Research Laboratory, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Siever, Jodi E. [Department of Biostatistics, Public Health Innovation and Decision Support Population and Public Health, Alberta Health Services, Calgary, Alberta (Canada)] [Department of Biostatistics, Public Health Innovation and Decision Support Population and Public Health, Alberta Health Services, Calgary, Alberta (Canada); Thomson, Thomas A. [Department of Pathology, British Columbia Cancer Agency–Vancouver, Vancouver, British Columbia (Canada) [Department of Pathology, British Columbia Cancer Agency–Vancouver, Vancouver, British Columbia (Canada); Faculty of Medicine, University of British Columbia, Vancouver, British Columbia (Canada); Magliocco, Anthony M. [Department of Pathology, Tom Baker Cancer Centre, Calgary, Alberta (Canada) [Department of Pathology, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Faculty of Medicine, University of Calgary, Calgary, Alberta (Canada); Hao, Desirée, E-mail: Desiree.Hao@albertahealthservices.ab.ca [Department of Medical Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada) [Department of Medical Oncology, Tom Baker Cancer Centre, Calgary, Alberta (Canada); Faculty of Medicine, University of Calgary, Calgary, Alberta (Canada)

    2013-04-01

    Purpose: We sought to evaluate the prognostic/predictive value of ERCC1 and XPF in patients with nonmetastatic nasopharyngeal carcinoma (NPC) treated with curative intent. Methods and Materials: ERCC1 and XPF protein expression was evaluated by immunofluorescence combined with automated quantitative analysis (AQUA) using the FL297 and 3F2 antibodies, respectively. ERCC1 and XPF protein expression levels were correlated with clinical outcomes. Results: Patient characteristics were as follows: mean age 52 years (range, 18-85 years), 67% male, 72% Karnofsky performance status (KPS) ?90%, World Health Organization (WHO) type 1/2/3 = 12%/28%/60%, stage III/IV 65%. With a median follow-up time of 50 months (range, 2.9 to 120 months), the 5-year overall survival (OS) was 70.8%. Median standardized nuclear AQUA scores were used as cutpoints for ERCC1 (n=138) and XPF (n=130) protein expression. Agreement between dichotomized ERCC1 and XPF scores was high at 79.4% (kappa = 0.587, P<.001). Neither biomarker predicted locoregional recurrence, DFS, or OS after adjustment for age and KPS, irrespective of stratification by stage, WHO type, or treatment. Conclusions: Neither ERCC1 nor XPF, analyzed by quantitative immunohistochemistry using the FL297 and 3F2 antibodies, was prognostic or predictive in this cohort of NPC patients.

  5. Copolymers For Capillary Gel Electrophoresis

    DOEpatents

    Liu, Changsheng (State College, PA); Li, Qingbo (State College, PA)

    2005-08-09

    This invention relates to an electrophoresis separation medium having a gel matrix of at least one random, linear copolymer comprising a primary comonomer and at least one secondary comonomer, wherein the comonomers are randomly distributed along the copolymer chain. The primary comonomer is an acrylamide or an acrylamide derivative that provides the primary physical, chemical, and sieving properties of the gel matrix. The at least one secondary comonomer imparts an inherent physical, chemical, or sieving property to the copolymer chain. The primary and secondary comonomers are present in a ratio sufficient to induce desired properties that optimize electrophoresis performance. The invention also relates to a method of separating a mixture of biological molecules using this gel matrix, a method of preparing the novel electrophoresis separation medium, and a capillary tube filled with the electrophoresis separation medium.

  6. DNA typing by capillary electrophoresis

    SciTech Connect

    Zhang, N.

    1997-10-08

    Capillary electrophoresis is becoming more and more important in nucleic acid analysis including DNA sequencing, typing and disease gene measurements. This work summarized the background of DNA typing. The recent development of capillary electrophoresis was also discussed. The second part of the thesis showed the principle of DNA typing based on using the allelic ladder as the absolute standard ladder in capillary electrophoresis system. Future work will be focused on demonstrating DNA typing on multiplex loci and examples of disease diagnosis in the on-line format of PCR-CE. Also capillary array electrophoresis system should allow high throughput, fast speed DNA typing. Only the introduction and conclusions for this report are available here. A reprint was removed for separate processing.

  7. Capillary electrophoresis-mass spectrometry of carbohydrates

    PubMed Central

    Zaia, Joseph

    2014-01-01

    The development of methods for capillary electrophoresis (CE) with on-line mass spectrometric detection (CE/MS) is driven by the need for accurate, robust and sensitive glycomics analysis for basic biomedicine, biomarker discovery, and analysis of recombinant protein therapeutics. One important capability is to profile glycan mixtures with respect to the patterns of substituents including sialic acids, acetate, sulfate, phosphate, and other groups. There is additional need for an MS-compatible separation system capable of resolving carbohydrate isomers. This review summarizes applications of CS/MS to analysis of carbohydrates, glycoproteins and glycopeptides that have appeared since 2008. Readers are referred to recent comprehensive reviews covering earlier publications. PMID:23386333

  8. Cytokine Analysis by Immunoaffinity Capillary Electrophoresis

    PubMed Central

    Mendonca, Mark; Kalish, Heather

    2014-01-01

    Immunoaffinity capillary electrophoresis (ICE) is a powerful tool used to detect and quantify target proteins of interest in complex biological fluids. The target analyte is captured and bound to antibodies immobilized onto the wall of a capillary, labeled in situ with a fluorescent dye, eluted and detected online using laser-induced fluorescence following electrophoretic separation. Here, we illustrate how to construct an immunoaffinity capillary and utilize it to run ICE in order to capture and quantify target cytokines and chemokines from a clinical sample. PMID:22976107

  9. Quantitation of adsorption and conjugation of plasma proteins by residual glutaraldehyde in fixed collagenous tissue with radioiodinated plasma proteins.

    PubMed

    Setiawan, T; Dewanjee, M K; Gross, D R

    1998-01-01

    Residual glutaraldehyde (GA) in collagenous cardiovascular tissue prostheses after multiple saline rinses remains in the prostheses and accounts for adsorption and conjugation of a variety of plasma proteins. This may account for later beneficial or adverse effects. Human serum albumin (SA), gamma globulin (GG), and fibrinogen (FB) were iodinated with 125I using the iodogen-transfer technique. Bovine pericardium (PC) was fixed with 0.5% GA for 24 hr and rinsed to remove excess GA. Fresh and GA-fixed PC (FRPC, GAPC: 1 x 1 cm2), in triplicate, were incubated with 0.5-1.0 microCi of tracers in human, porcine, or bovine blood (2 ml) for a period of 0.5, 1, 2, and 3 hr and washed (5x) with saline. Maximum adsorbed proteins per unit weight of collagen (pmol/mg of PC, mean +/- SD) at 3 hr on FRPC and GAPC were quantified with a gamma counter. Fixed PC absorbed significantly more plasma proteins from blood than fresh PC. These conjugated plasma proteins are tightly bound to fixed PC. The adsorbed and conjugated plasma proteins for GAPC and FRPC have the same sequence: SA > GG > FB vs SA > GG > FB. Protein conjugation may affect the remodeling of collagenous cardiovascular tissue prostheses post implantation. PMID:9804469

  10. Label-Free Quantitative Mass Spectrometry Reveals a Panel of Differentially Expressed Proteins in Colorectal Cancer

    PubMed Central

    Fan, Nai-Jun; Gao, Jiang-Ling; Liu, Yan; Song, Wei; Zhang, Zhan-Yang; Gao, Chun-Fang

    2015-01-01

    To identify potential biomarkers involved in CRC, a shotgun proteomic method was applied to identify soluble proteins in three CRCs and matched normal mucosal tissues using high-performance liquid chromatography and mass spectrometry. Label-free protein profiling of three CRCs and matched normal mucosal tissues were then conducted to quantify and compare proteins. Results showed that 67 of the 784 identified proteins were linked to CRC (28 upregulated and 39 downregulated). Gene Ontology and DAVID databases were searched to identify the location and function of differential proteins that were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, and so on. Among the differentially expressed proteins, tropomyosin-3 (TPM3), endoplasmic reticulum resident protein 29 (ERp29), 18?kDa cationic antimicrobial protein (CAMP), and heat shock 70?kDa protein 8 (HSPA8) were verified to be upregulated in CRC tissue and seven cell lines through western blot analysis. Furthermore, the upregulation of TPM3, ERp29, CAMP, and HSPA8 was validated in 69 CRCs byimmunohistochemistry (IHC) analysis. Combination of TPM3, ERp29, CAMP, and HSPA8 can identify CRC from matched normal mucosal achieving an accuracy of 73.2% using IHC score. These results suggest that TPM3, ERp29, CAMP, and HSPA8 are great potential IHC diagnostic biomarkers for CRC. PMID:25699276

  11. Label-free quantitative mass spectrometry reveals a panel of differentially expressed proteins in colorectal cancer.

    PubMed

    Fan, Nai-Jun; Gao, Jiang-Ling; Liu, Yan; Song, Wei; Zhang, Zhan-Yang; Gao, Chun-Fang

    2015-01-01

    To identify potential biomarkers involved in CRC, a shotgun proteomic method was applied to identify soluble proteins in three CRCs and matched normal mucosal tissues using high-performance liquid chromatography and mass spectrometry. Label-free protein profiling of three CRCs and matched normal mucosal tissues were then conducted to quantify and compare proteins. Results showed that 67 of the 784 identified proteins were linked to CRC (28 upregulated and 39 downregulated). Gene Ontology and DAVID databases were searched to identify the location and function of differential proteins that were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, and so on. Among the differentially expressed proteins, tropomyosin-3 (TPM3), endoplasmic reticulum resident protein 29 (ERp29), 18?kDa cationic antimicrobial protein (CAMP), and heat shock 70?kDa protein 8 (HSPA8) were verified to be upregulated in CRC tissue and seven cell lines through western blot analysis. Furthermore, the upregulation of TPM3, ERp29, CAMP, and HSPA8 was validated in 69 CRCs byimmunohistochemistry (IHC) analysis. Combination of TPM3, ERp29, CAMP, and HSPA8 can identify CRC from matched normal mucosal achieving an accuracy of 73.2% using IHC score. These results suggest that TPM3, ERp29, CAMP, and HSPA8 are great potential IHC diagnostic biomarkers for CRC. PMID:25699276

  12. Comparison of the capabilities of liquid isoelectric focusing–one-dimensional nonporous silica reversed-phase liquid chromatography–electrospray ionization time-of-flight mass spectrometry and liquid isoelectric focusing–one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis mass mapping for the analysis of intact protein molecular masses

    Microsoft Academic Search

    Daniel B Wall; Stephen J Parus; David M Lubman

    2001-01-01

    Nonporous silica reversed-phase HPLC coupled to electrospray ionization with on-line time-of-flight mass spectrometric detection (NPS-RP-HPLC–ESI-TOF-MS) is shown to be an effective liquid phase method for obtaining the molecular masses of proteins from pH fractionated cellular lysates where the method is capable of generating the same banding patterns typically observed using gel phase one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The liquid-phase

  13. Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross-talk between excess zinc and iron deficiency.

    PubMed

    Zargar, Sajad Majeed; Kurata, Rie; Inaba, Shoko; Oikawa, Akira; Fukui, Risa; Ogata, Yoshiyuki; Agrawal, Ganesh Kumar; Rakwal, Randeep; Fukao, Yoichiro

    2015-04-01

    Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe-deficient plants. To understand cross-talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ-OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe-deficient media compared to basal MGRL solid medium. iTRAQ-OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross-talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe-deficient conditions. Plants grown on Fe-deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization. PMID:25641898

  14. iTRAQ-Based Quantitative Proteomic Analysis on S100 Calcium Binding Protein A2 in Metastasis of Laryngeal Cancer

    PubMed Central

    Zha, Cong; Jiang, Xue Hua; Peng, Shi Fang

    2015-01-01

    Laryngeal cancer is the most frequent neoplasm in the head and neck region, with the vast majority of tumors originating from squamous cells. The survival rate of patients with laryngeal cancer has not improved substantially over the past 25 years. To acquire further knowledge regarding the molecules responsible for laryngeal cancer oncogenesis and, in turn, to improve target therapy?iTRAQ and mass spectrometry analysis were utilized to detect differences in protein expression from 15 paired laryngeal cancer and adjacent non-cancerous tissue samples. Using mass spectrometry analysis, the expression levels of 100 proteins in laryngeal cancer samples were distinct from the non-tumor, non-cancerous samples. Further validation of the differentially expressed proteins S100A2, KRT16, FGB and HSPB1 were carried out using quantitative real-time RT-PCR, immunoblot and immunohistochemistry. Functional analysis of one of the highly expressed proteins, S100 calcium binding protein A2 (S100A2), was performed using RNA interference. As a consequence, attenuated S100A2 expression enhanced the ability of HEp-2 cell lines to migrate and invade in vitro. Our investigation complements the current understanding of laryngeal cancer progression. Furthermore, this study supports the concept that enhanced expression of S100A2 may be a promising strategy in developing novel cancer therapeutic drugs. PMID:25874882

  15. Quantitation of low concentrations of polysorbates in high protein concentration formulations by solid phase extraction and cobalt-thiocyanate derivatization.

    PubMed

    Kim, Justin; Qiu, Jinshu

    2014-01-01

    A spectrophotometric method was developed to quantify low polysorbate (PS) levels in biopharmaceutical formulations containing high protein concentrations. In the method, Oasis HLB solid phase extraction (SPE) cartridge was used to extract PS from high protein concentration formulations. After loading a sample, the cartridge was washed with 4M guanidine HCl and 10% (v/v) methanol, and the retained PS was eluted by acetonitrile. Following the evaporation of acetonitrile, aqueous cobalt-thiocyanate reagent was added to react with the polyoxyethylene oxide chain of polysorbates to form a blue colored PS-cobaltothiocyante complex. This colored complex was then extracted into methylene chloride and measured spectrophotometrically at 620 nm. The method performance was evaluated on three products containing 30-40 mg L(-1) PS-20 and PS-80 in ?70 g L(-1) protein formulations. The method was specific (no matrix interference identified in three types of protein formulations), sensitive (quantitation limit of 10 mg L(-1) PS) and robust with good precision (relative standard deviation ?6.4%) and accuracy (spike recoveries from 95% to 101%). The linear range of the method for both PS-20 and PS-80 was 10 to 80 mg L(-1) PS. By diluting samples with 6M guanidine HCl and/or using different methylene chloride volumes to extract the colored complexes of standards and samples, the method could accurately and precisely quantify 40 mg L(-1) PS in up to 300 g L(-1) protein formulations. PMID:24331050

  16. Quantitative analysis of the mitochondrial and plastid proteomes of the moss Physcomitrella patens reveals protein macrocompartmentation and microcompartmentation.

    PubMed

    Mueller, Stefanie J; Lang, Daniel; Hoernstein, Sebastian N W; Lang, Erika G E; Schuessele, Christian; Schmidt, Anton; Fluck, Melanie; Leisibach, Desirée; Niegl, Christina; Zimmer, Andreas D; Schlosser, Andreas; Reski, Ralf

    2014-04-01

    Extant eukaryotes are highly compartmentalized and have integrated endosymbionts as organelles, namely mitochondria and plastids in plants. During evolution, organellar proteomes are modified by gene gain and loss, by gene subfunctionalization and neofunctionalization, and by changes in protein targeting. To date, proteomics data for plastids and mitochondria are available for only a few plant model species, and evolutionary analyses of high-throughput data are scarce. We combined quantitative proteomics, cross-species comparative analysis of metabolic pathways, and localizations by fluorescent proteins in the model plant Physcomitrella patens in order to assess evolutionary changes in mitochondrial and plastid proteomes. This study implements data-mining methodology to classify and reliably reconstruct subcellular proteomes, to map metabolic pathways, and to study the effects of postendosymbiotic evolution on organellar pathway partitioning. Our results indicate that, although plant morphologies changed substantially during plant evolution, metabolic integration of organelles is largely conserved, with exceptions in amino acid and carbon metabolism. Retargeting or regulatory subfunctionalization are common in the studied nucleus-encoded gene families of organelle-targeted proteins. Moreover, complementing the proteomic analysis, fluorescent protein fusions revealed novel proteins at organelle interfaces such as plastid stromules (stroma-filled tubules) and highlight microcompartments as well as intercellular and intracellular heterogeneity of mitochondria and plastids. Thus, we establish a comprehensive data set for mitochondrial and plastid proteomes in moss, present a novel multilevel approach to organelle biology in plants, and place our findings into an evolutionary context. PMID:24515833

  17. DESIGN OF SRM BASED MASS SPECTROMETRY METHODS FOR QUANTITATIVE ANALYSES OF PORCINE ACUTE PHASE PROTEINS

    E-print Network

    Schierup, Mikkel Heide

    DESIGN OF SRM BASED MASS SPECTROMETRY METHODS FOR QUANTITATIVE ANALYSES OF PORCINE ACUTE PHASE for the quantification of four porcine APPs: Hp, ITIH4, Apo A-I and fetuin A. The measurement of these four APPs (intense transitions); B) Bad proteotypic peptide (non-intense transitions) 2) Quantification of porcine

  18. Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader.

    PubMed

    Duellman, Tyler; Burnett, John; Yang, Jay

    2015-03-15

    Traditional assays for secreted proteins include methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) detection of the protein in the cell culture medium. We describe a method for the detection of a secreted protein based on fluorescent measurement of an mCherry fusion reporter. This microplate reader-based mCherry fluorescence detection method has a wide dynamic range of 4.5 orders of magnitude and a sensitivity that allows detection of 1 to 2fmol fusion protein. Comparison with the Western blot detection method indicated greater linearity, wider dynamic range, and a similar lower detection threshold for the microplate-based fluorescent detection assay of secreted fusion proteins. An mCherry fusion protein of matrix metalloproteinase-9 (MMP-9), a secreted glycoprotein, was created and expressed by transfection of human embryonic kidney (HEK) 293 cells. The cell culture medium was assayed for the presence of the fluorescent signal up to 32h after transfection. The secreted MMP-9-mCherry fusion protein was detected 6h after transfection with a linear increase in signal intensity over time. Treatment with chloroquine, a drug known to inhibit the secretion of many proteins, abolished the MMP-9-mCherry secretion, demonstrating the utility of this method in a biological experiment. PMID:25542417

  19. Quantitative proteomics reveals the effect of protein glycosylation in soybean root under flooding stress

    PubMed Central

    Mustafa, Ghazala; Komatsu, Setsuko

    2014-01-01

    Flooding stress has a negative impact on soybean cultivation because it severely impairs growth and development. To understand the flooding responsive mechanism in early stage soybeans, a glycoproteomic technique was used. Two-day-old soybeans were treated with flooding for 2 days and roots were collected. Globally, the accumulation level of glycoproteins, as revealed by cross-reaction with concanavalin A decreased by 2 days of flooding stress. Glycoproteins were enriched from total protein extracts using concanavalin A lectin resin and analyzed using a gel-free proteomic technique. One-hundred eleven and 69 glycoproteins were identified without and with 2 days of flooding stress, respectively. Functional categorization of these identified glycoproteins indicated that the accumulation level of proteins related to protein degradation, cell wall, and glycolysis increased, while stress-related proteins decreased under flooding stress. Also the accumulation level of glycoproteins localized in the secretory pathway decreased under flooding stress. Out of 23 common glycoproteins between control and flooding conditions, peroxidases and glycosyl hydrolases were decreased by 2 days of flooding stress. mRNA expression levels of proteins in the endoplasmic reticulum and N-glycosylation related proteins were downregulated by flooding stress. These results suggest that flooding might negatively affect the process of N-glycosylation of proteins related to stress and protein degradation; however glycoproteins involved in glycolysis are activated. PMID:25477889

  20. A Quantitative Microscopy Technique for Determining the Number of Specific Proteins in Cellular Compartments

    PubMed Central

    Mutch, Sarah A.; Gadd, Jennifer C.; Fujimoto, Bryant S.; Kensel-Hammes, Patricia; Schiro, Perry G.; Bajjalieh, Sandra M.; Chiu, Daniel T.

    2013-01-01

    This protocol describes a method to determine both the average number and variance of proteins in the few to tens of copies in isolated cellular compartments, such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number but lack information on the variance or are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling the cellular compartment with fluorescent primary-secondary antibody complexes, TIRF (total internal reflection fluorescence) microscopy imaging of the cellular compartment, digital image analysis, and deconvolution of the fluorescence intensity data. A minimum of 2.5 days is required to complete the labeling, imaging, and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes. PMID:22094731

  1. POTENTIALS AND METHOD IMPROVEMENTS OF CAPILLARY ZONE ELECTROPHORESIS FOR USE IN SPELT BREEDING PROGRAMS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Capillary zone electrophoresis (CZE) in acidic buffer systems is capable of separating cereal storage proteins based on similar separation principles as classical acidic polyacrylamide gel electrophoresis. However, it is faster, its resolution is distinctly higher and data evaluation is much simpler...

  2. Potentials and Method Improvements of Capillary Zone Electrophoresis for Use in Spelt Breeding Programs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Capillary zone electrophoresis (CZE) in acidic buffer systems is capable of separating cereal storage proteins based on similar separation principles as classical acidic polyacrylamide gel electrophoresis. However, it is faster, its resolution is distinctly higher and data evaluation is much simpler...

  3. Simulation of Two Dimensional Electrophoresis and Tandem Mass Spectrometry for Teaching Proteomics

    ERIC Educational Resources Information Center

    Fisher, Amanda; Sekera, Emily; Payne, Jill; Craig, Paul

    2012-01-01

    In proteomics, complex mixtures of proteins are separated (usually by chromatography or electrophoresis) and identified by mass spectrometry. We have created 2DE Tandem MS, a computer program designed for use in the biochemistry, proteomics, or bioinformatics classroom. It contains two simulations--2D electrophoresis and tandem mass spectrometry.…

  4. Quantitative Proteomic Analysis in Metastatic Renal Cell Carcinoma Reveals a Unique Set of Proteins with Potential Prognostic Significance*

    PubMed Central

    Masui, Olena; White, Nicole M. A.; DeSouza, Leroi V.; Krakovska, Olga; Matta, Ajay; Metias, Shereen; Khalil, Bishoy; Romaschin, Alexander D.; Honey, R. John; Stewart, Robert; Pace, Kenneth; Bjarnason, Georg A.; Siu, K. W. Michael; Yousef, George M.

    2013-01-01

    Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies, and patients have a dismal prognosis, with a <10% five-year survival rate. The identification of markers that can predict the potential for metastases will have a great effect in improving patient outcomes. In this study, we used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to identify proteins that are differentially expressed in metastatic and primary RCC. We identified 1256 non-redundant proteins, and 456 of these were quantified. Further analysis identified 29 proteins that were differentially expressed (12 overexpressed and 17 underexpressed) in metastatic and primary RCC. Dysregulated protein expressions of profilin-1 (Pfn1), 14–3-3 zeta/delta (14–3-3?), and galectin-1 (Gal-1) were verified on two independent sets of tissues by means of Western blot and immunohistochemical analysis. Hierarchical clustering analysis showed that the protein expression profile specific for metastatic RCC can distinguish between aggressive and non-aggressive RCC. Pathway analysis showed that dysregulated proteins are involved in cellular processes related to tumor progression and metastasis. Furthermore, preliminary analysis using a small set of tumors showed that increased expression of Pfn1 is associated with poor outcome and is a potential prognostic marker in RCC. In addition, 14–3-3? and Gal-1 also showed higher expression in tumors with poor prognosis than in those with good prognosis. Dysregulated proteins in metastatic RCC represent potential prognostic markers for kidney cancer patients, and a greater understanding of their involved biological pathways can serve as the foundation of the development of novel targeted therapies for metastatic RCC. PMID:23082029

  5. Quantitative Proteomic Analysis of Cellular Protein Modulation upon Inhibition of the NEDD8-Activating Enzyme by MLN4924

    PubMed Central

    Liao, Hua; Liu, Xiaozhen J.; Blank, Jonathan L.; Bouck, David C.; Bernard, Hugues; Garcia, Khristofer; Lightcap, Eric S.

    2011-01-01

    Cullin-RING ubiquitin ligases (CRLs) are responsible for the ubiquitination of many cellular proteins, thereby targeting them for proteasomal degradation. In most cases the substrates of the CRLs have not been identified, although many of those that are known have cancer relevance. MLN4924, an investigational small molecule that is a potent and selective inhibitor of the Nedd8-activating enzyme (NAE), is currently being explored in Phase I clinical trials. Inhibition of Nedd8-activating enzyme by MLN4924 prevents the conjugation of cullin proteins with NEDD8, resulting in inactivation of the entire family of CRLs. We have performed stable isotope labeling with amino acids in cell culture analysis of A375 melanoma cells treated with MLN4924 to identify new CRL substrates, confidently identifying and quantitating 5122–6012 proteins per time point. Proteins such as MLX, EID1, KLF5, ORC6L, MAGEA6, MORF4L2, MRFAP1, MORF4L1, and TAX1BP1 are rapidly stabilized by MLN4924, suggesting that they are novel CRL substrates. Proteins up-regulated at later times were also identified and siRNA against their corresponding genes were used to evaluate their influence on MLN4924-induced cell death. Thirty-eight proteins were identified as being particularly important for the cytotoxicity of MLN4924. Strikingly, these proteins had roles in cell cycle, DNA damage repair, and ubiquitin transfer. Therefore, the combination of RNAi with stable isotope labeling with amino acids in cell culture provides a paradigm for understanding the mechanism of action of novel agents affecting the ubiquitin proteasome system and a path to identifying mechanistic biomarkers. PMID:21873567

  6. A quantitative model of odor deactivation based on the redox shift of the pheromone-binding protein im moth antennae.

    PubMed

    Kaissling, K E

    1998-11-30

    Recent in vitro experiments with homogenates of isolated olfactory hairs of Antheraea polyphemus suggest that the pheromone-binding protein (PBP) is involved not only in pheromone solubilization and transport but also in pheromone deactivation. PBP occurs in a reduced form with one or two disulfide bridges (PBP(red)) and in the oxidized form with three bridges (PBP(ox)). From kinetic experiments it was concluded that the pheromone is first bound to PBP(red). This complex activates the receptor molecules and then turns into the oxidized form which--according to our working hypothesis--is unable to activate further receptor molecules. Apparently, the pheromone bound to the PBP (both forms) is protected from enzymatic degradation into nonexcitatory metabolites. A quantitative kinetic model of pheromone deactivation was developed (in collaboration with J. Thorson, Oxford) in which the receptor molecules are considered to act as enzymes catalyzing the redox shift of the binding protein. PMID:10049225

  7. Metabolic labeling of plant cell cultures with K15NO3 as a tool for quantitative analysis of proteins and metabolites

    Microsoft Academic Search

    Wolfgang R Engelsberger; Alexander Erban; Joachim Kopka; Waltraud X Schulze

    2006-01-01

    Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that address biological questions beyond the establishment of protein lists. Here, we propose the use of 15N-KNO3 as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population. Proteins from such metabolically labeled culture are distinguishable from

  8. Analysis of protein turnover by quantitative SNAP-based pulse-chase imaging.

    PubMed

    Bodor, Dani L; Rodríguez, Mariluz Gómez; Moreno, Nuno; Jansen, Lars E T

    2012-06-01

    Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP-tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse-chase and quench-chase-pulse experiments. These time-slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP-tagging in fixed- and live-cell studies and evaluate the recently developed fast-acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification. PMID:23129118

  9. Quantitative Proteomic Profiling Reveals Differentially Regulated Proteins in Cystic Fibrosis Cells

    PubMed Central

    Rauniyar, Navin; Gupta, Vijay; Balch, William E.; Yates, John R.

    2014-01-01

    The most prevalent cause of cystic fibrosis (CF) is the deletion of a phenylalanine residue at position 508 in CFTR (?F508-CFTR) protein. The mutated protein fails to fold properly, is retained in the endoplasmic reticulum via the action of molecular chaperones, and is tagged for degradation. In this study, the differences in protein expression levels in CF cell models were assessed using a systems biology approach aided by the sensitivity of MudPIT proteomics. Analysis of the differential proteome modulation without a priori hypotheses has the potential to identify markers that have not yet been documented. These may also serve as the basis for developing new diagnostic and treatment modalities for CF. Several novel differentially expressed proteins observed in our study are likely to play important roles in the pathogenesis of CF and may serve as a useful resource for the CF scientific community. PMID:24818864

  10. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

    NASA Astrophysics Data System (ADS)

    Sun, Liangliang; Bertke, Michelle M.; Champion, Matthew M.; Zhu, Guijie; Huber, Paul W.; Dovichi, Norman J.

    2014-03-01

    While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development.

  11. Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

    PubMed Central

    Sun, Liangliang; Bertke, Michelle M.; Champion, Matthew M.; Zhu, Guijie; Huber, Paul W.; Dovichi, Norman J.

    2014-01-01

    While there is a rich literature on transcription dynamics during the development of many organisms, protein data is limited. We used iTRAQ isotopic labeling and mass spectrometry to generate the largest developmental proteomic dataset for any animal. Expression dynamics of nearly 4,000 proteins of Xenopus laevis was generated from fertilized egg to neurula embryo. Expression clusters into groups. The cluster profiles accurately reflect the major events that mark changes in gene expression patterns during early Xenopus development. We observed decline in the expression of ten DNA replication factors after the midblastula transition (MBT), including a marked decline of the licensing factor XCdc6. Ectopic expression of XCdc6 leads to apoptosis; temporal changes in this protein are critical for proper development. Measurement of expression in single embryos provided no evidence for significant protein heterogeneity between embryos at the same stage of development. PMID:24626130

  12. Quantitative proteomic profiling reveals differentially regulated proteins in cystic fibrosis cells.

    PubMed

    Rauniyar, Navin; Gupta, Vijay; Balch, William E; Yates, John R

    2014-11-01

    The most prevalent cause of cystic fibrosis (CF) is the deletion of a phenylalanine residue at position 508 in CFTR (?F508-CFTR) protein. The mutated protein fails to fold properly, is retained in the endoplasmic reticulum via the action of molecular chaperones, and is tagged for degradation. In this study, the differences in protein expression levels in CF cell models were assessed using a systems biology approach aided by the sensitivity of MudPIT proteomics. Analysis of the differential proteome modulation without a priori hypotheses has the potential to identify markers that have not yet been documented. These may also serve as the basis for developing new diagnostic and treatment modalities for CF. Several novel differentially expressed proteins observed in our study are likely to play important roles in the pathogenesis of CF and may serve as a useful resource for the CF scientific community. PMID:24818864

  13. Changes in protein expression in maturing equine testis: a quantitative DIGE analysis

    E-print Network

    Roper-Foo, Pilar

    2011-01-11

    . The light regions are associated with localized initiation and regulation of spermatogenesis. It is hypothesized that different proteins and protein expression levels in the light regions are factors contributing to successful sperm production..., macrophages and small seminiferous tubules that are not producing sperm. In contrast, the light tissue is composed of fewer Leydig cells and macrophages as the seminiferous tubules (containing non-pigmented cells) increase in size to occupy a larger...

  14. Quantitative assay of S-100 protein in mouse brain cortex synaptosomes

    Microsoft Academic Search

    M. V. Starostina; A. A. Nikolaenkova; T. K. Malup; L. I. Korochkin; S. M. Sviridov

    1993-01-01

    1.Data on the presence of S-100 protein in synaptic endings are revised, and evidence is given in favor of its localization inside mouse brain cortex synaptosomes and on the surface of their external membrane.2.For identification of the S-100-specific polypeptide, proteins of external synaptosomal membranes were iodinated with lactoperoxidase fixed on cyanogen bromide (CNBr)-Sepharose, and after synaptosome lysis S-100-positive material was

  15. Inhibition of Bacterial Conjugation by Phage M13 and Its Protein g3p: Quantitative Analysis and Model

    PubMed Central

    Lin, Abraham; Jimenez, Jose; Derr, Julien; Vera, Pedro; Manapat, Michael L.; Esvelt, Kevin M.; Villanueva, Laura; Liu, David R.; Chen, Irene A.

    2011-01-01

    Conjugation is the main mode of horizontal gene transfer that spreads antibiotic resistance among bacteria. Strategies for inhibiting conjugation may be useful for preserving the effectiveness of antibiotics and preventing the emergence of bacterial strains with multiple resistances. Filamentous bacteriophages were first observed to inhibit conjugation several decades ago. Here we investigate the mechanism of inhibition and find that the primary effect on conjugation is occlusion of the conjugative pilus by phage particles. This interaction is mediated primarily by phage coat protein g3p, and exogenous addition of the soluble fragment of g3p inhibited conjugation at low nanomolar concentrations. Our data are quantitatively consistent with a simple model in which association between the pili and phage particles or g3p prevents transmission of an F plasmid encoding tetracycline resistance. We also observe a decrease in the donor ability of infected cells, which is quantitatively consistent with a reduction in pili elaboration. Since many antibiotic-resistance factors confer susceptibility to phage infection through expression of conjugative pili (the receptor for filamentous phage), these results suggest that phage may be a source of soluble proteins that slow the spread of antibiotic resistance genes. PMID:21637841

  16. Silver Staining of 2D Electrophoresis Gels Thierry Rabilloud

    E-print Network

    Paris-Sud XI, Université de

    Silver Staining of 2D Electrophoresis Gels Thierry Rabilloud CEA-DSV-iRTSV/LCBM and UMR CNRS separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use spectrometry, quantification, polyacrylamide gels, protein visualisation, silver staining #12;1. Introduction

  17. Original article Two-dimensional gel electrophoresis of membrane

    E-print Network

    Paris-Sud XI, Université de

    Original article Two-dimensional gel electrophoresis of membrane proteins from ectomycorrhizal-dimensional polyacrylamide gels. Gels with limited back- ground staining and streaking and with clearly efficacité et leur compatibilité avec l'obtention de gels d'électro- phorèse bidimensionnelle. Une fraction

  18. Simple radiometric method for accurately quantitating epitope densities of hapten-protein conjugates with sulfhydryl linkages.

    PubMed

    Peterson, Eric C; Hambuchen, Michael D; Tawney, Rachel L; Gunnell, Melinda G; Cowell, James L; Lay, Jackson O; Blough, Bruce E; Carroll, F Ivy; Owens, S Michael

    2014-12-17

    Control of small molecule hapten epitope densities on antigenic carrier proteins is essential for development and testing of optimal conditions for vaccines. Yet, accurate determination of epitope density can be extremely difficult to accomplish, especially with the use of small haptens, large molecular weight carrier proteins, and limited amounts of protein. Here we report a simple radiometric method that uses (14)C-labeled cystine to measure hapten epitope densities during sulfhydryl conjugation of haptens to maleimide activated carrier proteins. The method was developed using a (+)-methamphetamine (METH)-like hapten with a sulfhydryl terminus, and two prototype maleimide activated carrier proteins, bovine serum albumin (BSA) and immunocyanin monomers of keyhole limpet hemocyanin. The method was validated by immunochemical analysis of the hapten-BSA conjugates, and least-squares linear regression analysis of epitope density values determined by the new radiometric method versus values determined by matrix-assisted laser desorption/ionization mass spectrometry. Results showed that radiometric epitope density values correlated extremely well with the mass spectrometrically derived values (r(2) = 0.98, y = 0.98x + 0.91). This convenient and simple method could be useful during several stages of vaccine development including the optimization and monitoring of conditions for hapten-protein conjugations, and choosing the most effective epitope densities for conjugate vaccines. PMID:25426820

  19. Patterned library analysis: A method for the quantitative assessment of hypotheses concerning the determinants of protein structure

    PubMed Central

    Lahr, Steven J.; Broadwater, Anne; Carter, Charles W.; Collier, Martha L.; Hensley, Lucinda; Waldner, Jennifer C.; Pielak, Gary J.; Edgell, Marshall Hall

    1999-01-01

    Site-directed mutagenesis and combinatorial libraries are powerful tools for providing information about the relationship between protein sequence and structure. Here we report two extensions that expand the utility of combinatorial mutagenesis for the quantitative assessment of hypotheses about the determinants of protein structure. First, we show that resin-splitting technology, which allows the construction of arbitrarily complex libraries of degenerate oligonucleotides, can be used to construct more complex protein libraries for hypothesis testing than can be constructed from oligonucleotides limited to degenerate codons. Second, using eglin c as a model protein, we show that regression analysis of activity scores from library data can be used to assess the relative contributions to the specific activity of the amino acids that were varied in the library. The regression parameters derived from the analysis of a 455-member sample from a library wherein four solvent-exposed sites in an ?-helix can contain any of nine different amino acids are highly correlated (P < 0.0001, R2 = 0.97) to the relative helix propensities for those amino acids, as estimated by a variety of biophysical and computational techniques. PMID:10611303

  20. Filamin C, a dysregulated protein in cancer revealed by label-free quantitative proteomic analyses of human gastric cancer cells

    PubMed Central

    Xu, Lei-Lei; Chen, Si-Jie; Yao, Jun; Jiang, Ying-Hua; Peng, Gang; Fang, Cai-Yun

    2015-01-01

    Gastric cancer (GC) is the fourth and fifth most common cancer in men and women, respectively. We identified 2,750 proteins at false discovery rates of 1.3% (protein) and 0.03% (spectrum) by comparing the proteomic profiles of three GC and a normal gastric cell lines. Nine proteins were significantly dysregulated in all three GC cell lines, including filamin C, a muscle-specific filamin and a large actin-cross-linking protein. Downregulation of filamin C in GC cell lines and tissues were verified using quantitative PCR and immunohistochemistry. Data-mining using public microarray datasets shown that filamin C was significantly reduced in many human primary and metastasis cancers. Transient expression or silencing of filamin C affected the proliferation and colony formation of cancer cells. Silencing of endogenous filamin C enhanced cancer cell migration and invasion, whereas ectopic expression of filamin C had opposing effects. Silencing of filamin C increased the expression of matrix metallopeptidase 2 and improved the metastasis of prostate cancer in a zebrafish model. High filamin C associated with better prognosis of prostate cancer, leukemia and breast cancer patients. These findings establish a functional role of filamin C in human cancers and these data will be valuable for further study of its mechanisms. PMID:25577646