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  • 1
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Escherichia coli ; SWISS-2DPAGE database ; Immobilized pH gradient ; Sequence tag ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The combination of two-dimensional polyacrylamide gel electrophoresis (2-DPAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d-top.html. Here we report recent progress in the development of the E. coli SWISS-2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman “sequence tag” approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.
    Additional Material: 4 Ill.
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  • 2
    ISSN: 0173-0835
    Keywords: Immunobloting ; Two-dimensional polyacrylamide gel electrophoresis ; Immobilized pH gradient ; Protein identification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Cell dysfunction or dysregulation in cancer generally results from complex gene interactions, numerous cellular events and environmental influences which modify gene expression or post-translational protein modifications. Genetic analysis in itself cannot always predict or diagnose multigenic diseases. The major technical difficulty is thus to detect, identify and measure simultaneously the expression of several genes and the post-translational modifications of their products. In order to progress to this direction, this paper describes a simple immunoblot method using several monoclonal antibodies to simultaneously analyze oncogene expression and cell cycle specific checkpoints in patient solid biopsies and transformed cell lines. One mg of normal human liver biopsy and HEPG2 (hepatoblastoma-derived cell line) protein samples have been separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were stained with amido black, scanned and tested separately with the nine monoclonal antibodies p53, c-myc, PCNA, MEK1, pan-ras, Cip1, Cdc2, Kip1, and TCTP. The nine antibodies of interest were then combined to form a mixture, and simultaneously used as the primary antibodies. This antibody mixture simultaneously detected the nine proteins of interest or both samples and it demonstrated the extensive expression changes and the presence of various isoforms most likely due to post-translational modifications of gene products.
    Additional Material: 3 Ill.
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  • 3
    ISSN: 0173-0835
    Keywords: Apolipoprotein E ; Immobilized pH gradient ; Phenotyping ; Immunoblotting ; Two-dimensional polyacrylamide gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Apolipoprotein E (apo E) is a normal component of several classes of plasma lipoproteins. Apo E phenotypes are closely related to total cholesterol, low density lipoprotein (LDL)-cholesterol and apo B concentration. The apo E 2/2 phenotype is related to the type III hyperlipoproteinemia due to the defective binding of apo E-2 to the hepatic receptors. The apo E 4/4 phenotype has been reported to be present in most elderly people suffering from the Alzheimer disease, and is associated with increased risk of coronary heart disease and Creutzfeld-Jakob disease. Therefore, apo E phenotyping is essential. The method described here uses a precast immobilized pH gradient, avoids time-consuming separation of lipoproteins from plasma, needs no pretreatment with neuraminidase and involves highly sensitive enhanced chemiluminescence for visualization. Therefore it has many advantages over previously published methods.
    Additional Material: 2 Ill.
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  • 4
    ISSN: 0173-0835
    Keywords: Amniotic fluid ; Two-dimensional polyacrylamide gel electrophoresis ; Immobilized pH gradient ; Protein map ; Gel matching ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Using updated technical procedures (immobilized pH gradients for isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: IPG/SDS-PAGE) we provide a two-dimensional (2-D) map of amniotic fluid (AF) proteins. This map comprises over 800 silver-stained spots. Over 150 spots have been identified by matching on the net with human plasma and cerebrospinal fluid maps available from SWISS 2DPAGE database; several additional spots were assigned by immunoblotting and/or microanalytical techniques. This report details our investigation on AF proteins focusing on the 17th week of gestation, when AF is most commonly used for clinical evaluation of fetal disorders. As a whole, the map displays a number of potential markers for fetal development and for gestation abnormalities. The 2-D electrophoretic technique allows the monitoring of all these proteins at the same time along with additional spots that may prove of diagnostic significance.
    Additional Material: 3 Ill.
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  • 5
    ISSN: 0173-0835
    Keywords: Ductal breast carcinoma ; Breast biopsy ; Two-dimensional polyacrylamide gel electrophoresis ; Reference map ; Immobilized pH gradient ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Reference two-dimensional (2-D) gels are presented for human breast ductal carcinoma and histologically normal tissue. Whole biopsy fragments were analyzed, including epithelial and nonepithelial components. Thirty-five spots have been assigned by gel matching to the human liver SWISS-2DPAGE reference map and/or to the human primary keratinocyte IPG map from the Danish Center for Human Genome. N-terminal microsequencing was applied to confirm randomly chosen matching assignments and to identify six new spots. Protein expression profiles in ductal carcinoma and in normal breast tissue appeared to be similar, except for a pattern consisting of 32 spots, which were highly expressed in all carcinoma specimens, and less intense and occasionally undetectable in normal tissue. This difference was statistically significant. Assignment has been obtained for several spots, namely GRP94, GRP78, GRP75, mitochondrial HSP60, calreticulin, protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase, collagen-binding protein 2, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, thioredoxin, cytochrome c oxidase VA subunit, tubulin β isoform and macrophage migration inhibitory factor (MIF). The cancer- and tissue-specificity of the described pattern was assessed by matching to the Swiss-2DPAGE human liver, hepatoma, lymphoma, erythroleukemia reference maps. The pattern of 32 spots was found to be indicative of epithelial neoplasia.
    Additional Material: 3 Ill.
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  • 6
    ISSN: 0173-0835
    Keywords: Sample application ; Two-dimensional polyacrylamide gel electrophoresis ; Immobilized pH gradient ; Protein characterization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A simple and inexpensive methacrylate rehydration chamber was built to accomodate ten immobilized pH gradient (IPG) strips. In the chamber, entire IPG gels were used for sample application, with the protein entering the gels during their rehydration. For rehydration, commercially available or laboratory-made strips were positioned in the grooves with the gel in contact with 500 μL of sample for 6 h or overnight. This avoided the use of sample cups, eliminated precipitation at the sample application site, thus improving resolution over the entire pH range of the gels. It also allowed precise control of protein amounts and sample volumes loaded into the IPG gels, and also lowered costs of reagents during rehydration and equilibration owing to the reduced volumes. Up to 5 mg of protein can be loaded on wide IPG gels and up to 15 mg of some samples on narrow pH range gels.
    Additional Material: 4 Ill.
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