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  • Two-dimensional polyacrylamide gel electrophoresis  (58)
  • Wiley-Blackwell  (58)
  • Blackwell Publishing Ltd
  • Nature Publishing Group
  • 1995-1999  (58)
  • 1985-1989
  • 1950-1954
  • 1998  (58)
Collection
Keywords
Publisher
  • Wiley-Blackwell  (58)
  • Blackwell Publishing Ltd
  • Nature Publishing Group
Years
  • 1995-1999  (58)
  • 1985-1989
  • 1950-1954
Year
  • 1
    Electronic Resource
    Electronic Resource
    Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 837-844 
    Details
    ISSN: 0173-0835
    Keywords: Membrane proteins ; Solubility ; Two-dimensional polyacrylamide gel electrophoresis ; Proteome ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190539
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  • 2
    Electronic Resource
    Electronic Resource
    Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 918-927 
    Details
    ISSN: 0173-0835
    Keywords: Matrix assisted laser desorption/ionization-mass spectrometry ; Peptide mass fingerprinting ; Two-dimensional polyacrylamide gel electrophoresis ; Silver staining ; Protein sequence coverage ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: 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.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190607
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  • 3
    Electronic Resource
    Electronic Resource
    Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 758-760 
    Details
    ISSN: 0173-0835
    Keywords: Membrane solubilization ; Thiourea ; Two-dimensional polyacrylamide gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The separation of membrane proteins by high-resolution two-dimensional electrophoresis was carried out. At high loads, these proteins are prone to precipitation, resulting in poor resolution. It is shown here that the use of thiourea, previously described for focusing in immobilized pH gradients, can be extended to conventional isoelectric focusing. As thiourea inhibits acrylamide polymerization, a modified photopolymerization system must be used. These modifications result in higher solubility of proteins during IEF, thereby increasing the resolution and capacity of the two-dimensional gels.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190526
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  • 4
    Electronic Resource
    Electronic Resource
    Evidence of intra- and extracellular modifications of monoclonal IgG polypeptide chains generating charge heterogeneity (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 767-775 
    Details
    ISSN: 0173-0835
    Keywords: Charge shift ; Aging ; Monoclonal antibody ; Glycosylation ; Deamidation ; Two-dimensional polyacrylamide gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The heavy and light chains of IgG monoclonal antibodies (mAbs) can be shown to be heterogeneous, with respect to isoelectric points, when analyzed by two-dimensional electrophoresis (2-DE). The molecular basis for this charge heterogeneity has not been clearly defined but it has been suggested that it could be due, in part, to differences in glycosylation. To investigate this possibility we have compared the 2-DE pattern of glycosylated and aglycosylated forms of the mouse IgG1 mAb (1B7-11), produced in vitro in the presence and absence of tunicamycin. Charge heterogeneity was shown not to be a consequence of glycosylation status. Intracellular and secreted IgG mAbs were also analyzed to investigate the time course of change in charge properties of the heavy and light chains. The charge heterogeneity was found to be generated intracellularly, and alterations in charge properties could be induced during incubation under physiological conditions. Semilogarithmic plots of the density of the principal heavy and light chain spots against incubation time showed linear relationships, suggesting that the charge shifts result from a first-order reaction. The semilogarithmic plot for the light chain correlated well with the time after IgG synthesis. These results suggest that the charge heterogeneity of an IgG mAb is due to intra- and extracellular modifications of the polypeptide chains which reflect “aging” of antibody molecules.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190528
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  • 5
    Electronic Resource
    Electronic Resource
    Two-dimensional gel electrophoresis separation and N-terminal sequence analysis of proteins from Clostridium pasteurianum W5 (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 802-806 
    Details
    ISSN: 0173-0835
    Keywords: Clostridium ; Protein sequence ; Proteome ; Two-dimensional polyacrylamide gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Proteins from Clostridium pasteurianum were separated by two-dimensional gel electrophoresis. Amino-terminal sequence determination and sequence analysis allowed the identification of 20 proteins, while 11 protein sequences remained unidentified and one protein appeared to have a blocked amino terminus.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190533
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  • 6
    Electronic Resource
    Electronic Resource
    Two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 797-801 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Bull seminal plasma proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins has been established. About 250 spots were detected after silver staining and polypeptides from 24 spots have been N-terminally sequenced. Major proteins already described in bull seminal plasma, like PDC-109 and aSFP, have been located on the map; proteins not yet reported in male reproductive tracts have been evidenced; for some polypeptides showing a previously unknown N-terminal sequence, structural similarities with proteins described in other organisms have been found. A reference map of seminal plasma proteins could be useful in relating protein pattern changes to physiopathological events influencing the reproductive sphere.
    Additional Material: 1 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190532
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  • 7
    Electronic Resource
    Electronic Resource
    Actin-binding proteins in mouse C2 myoblasts and myotubes: A combination of affinity chromatography and two-dimensional gel electrophoresis (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 826-833 
    Details
    ISSN: 0173-0835
    Keywords: Actin ; Actin-binding proteins ; C2 myoblast ; C2 myotubes ; Divinylsulfone-activated agarose (Mini-Leak) ; Two-dimensional polyacrylamide gel electrophoresis ; Affinity chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: This paper analyzes proteins expressed in a mouse muscle precursor cell line (C2 myoblasts) and compares them with those observed in differentiated myotubes from the same cell line. We observed hundreds of proteins in myoblasts using IPG two-dimensional gel electrophoresis but this number is greatly reduced using Mini-Leak (divinylsulfone-activated agarose) affinity chromatography. Two kinds of affinity columns were prepared. One contained a chemically modified monomeric actin bound to the affinity matrix. The second matrix contained a high-affinity actin-binding protein (DNase I) which was bound to the actin Mini-Leak column to block specific sites on actin. Actin-binding proteins in homogenates of myoblasts or myotubes were passed through the affinity columns and eluted under high salt conditions. The Mini-Leak affinity medium itself appeared to have little ability to bind proteins. Our two-dimensional (2-D) gels identified a small number of proteins and we are currently focusing our attention on a particular protein spot which could correspond to cofilin. Comparison of myoblast and myotube proteins using affinity chromatography shows no qualitative, clearly identifiable differences but the analysis is still in progress. These findings are discussed in relation to reports in which the myoblast-myotube transformation was associated with the up-regulation or de novo synthesis of more than ten proteins.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190537
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  • 8
    Electronic Resource
    Electronic Resource
    Analyzing glycoproteins separated by two-dimensional gel electrophoresis (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 981-988 
    Details
    ISSN: 0173-0835
    Keywords: Glycoprotein ; Two-dimensional polyacrylamide gel electrophoresis ; α1-Antitrypsin ; α2-HS Glycoprotein ; Protein isoforms ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two-dimensional (2-D) electrophoresis is the preferred method for separating the glycoforms of proteins. The isoforms usually present as ‘trains’ of spots in the first dimension and may also differ in molecular weight. The primary goal for analyzing the carbohydrate content of glycoprotein spots is to understand the ‘rules’ which govern the migration of glycoproteins in 2-D electrophoresis. These rules can then be used to produce predictive vectors to interpret changes in glycosylation patterns. Techniques for the analysis of oligosaccharides released from glycoproteins which have been electroblotted to PVDF membrane after one-dimensional (1-D) and 2-D preparative gel electrophoresis are described. The oligosaccharides are removed enzymatically (PNGase F of N-linked oligosaccharides) or chemically (β-elimination of O-linked oligosaccharides) and separated by high performance anion exchange chromatography (HPAEC-PAD) and identified by electrospray ionization mass spectrometry (ESI-MS) or analyzed directly by ESI-MS. After enzymic removal of the N-linked oligosaccharides the protein spots can be further analyzed by Edman sequence tagging for identification and quantitation of the protein and by acid hydrolysis for monosaccharide analysis of the O-linked oligosaccharides. These approaches have been proved on 1-D PAGE electroblotted bovine fetuin and human glycophorin A and then used to analyze two abundant proteins which separate as glycoforms on 2-D PAGE preparative narrow range (pH 4.5-5.5) blots of human plasma: α2-HS glycoprotein (human fetuin) and α1-antitrypsin (α1-protease inhibitor). It is apparent that both the macroheterogeneity (site occupation) and microheterogeneity (diversity of structures) of the glycosylation contribute to the separation of protein isoforms in 2-D PAGE.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190613
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  • 9
    Electronic Resource
    Electronic Resource
    Two-dimensional electrophoresis of human placental mitochondria and protein identification by mass spectrometry: Toward a human mitochondrial proteome (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 1006-1014 
    Details
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Silver staining ; Mass spectrometry ; Proteome ; Mitochondria ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Owing to the complexity of higher eukaryotic cells, characterization of a complete proteome is likely to be difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and “physiology” of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting, but other methods were also used (N-terminal microsequencing, blotting). The optimization steps in two-dimensional (2-D) electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficienciesThis work can be found on the Internet at the following address: http://www-dsv.cea.fr/MitoPick/Default.html.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150190616
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  • 10
    Electronic Resource
    Electronic Resource
    Proteome analysis: Biological assay or data archive? (1998)
    Weinheim : Wiley-Blackwell
    Electrophoresis 19 (1998), S. 1862-1871 
    Details
    ISSN: 0173-0835
    Keywords: Proteome ; Two-dimensional polyacrylamide gel electrophoresis ; Tandem mass spectrometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: In this review we examine the current state of proteome analysis. There are three main issues discussed: why it is necessary to study proteomes; how proteomes can be analyzed with current technology; and how proteome analysis can be used to enhance biological research. We conclude that proteome analysis is an essential tool in the understanding of regulated biological systems. Current technology, while still mostly limited to the more abundant proteins, enables the use of proteome analysis both to establish databases of proteins present, and to perform biological assays involving measurement of multiple variables. We believe that the utility of proteome analysis in future biological research will continue to be enhanced by further improvements in analytical technology.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150191104
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