Topic
Gel electrophoresis
About: Gel electrophoresis is a research topic. Over the lifetime, 26026 publications have been published within this topic receiving 1113565 citations.
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TL;DR: The hypothesis that an appropriate system of mildly denaturing solvents can amplify the tendency of single-base mismatches to produce conformational changes, such as bends in the double helix, and thereby increase the differential migration of DNA heterod uplexes and homoduplexes during gel electrophoresis is tested.
Abstract: Several techniques have recently been developed to detect single-base mismatches in DNA heteroduplexes that contain one strand of wild-type and one strand of mutated DNA. Here we tested the hypothesis that an appropriate system of mildly denaturing solvents can amplify the tendency of single-base mismatches to produce conformational changes, such as bends in the double helix, and thereby increase the differential migration of DNA heteroduplexes and homoduplexes during gel electrophoresis. The best separations of heteroduplexes and homoduplexes were obtained with a standard 6% polyacrylamide gel polymerized in 10% ethylene glycol/15% formamide/Tris-taurine buffer. As predicted by the hypothesis of solvent-induced bends, when the concentration of either ethylene glycol or formamide was increased, the differential migration decreased. Also, single-base mismatches within 50 bp of one end of a heteroduplex did not produce differential migration. Sixty of 68 single-base mismatches in a series of PCR products were detected in some 59 different sequence contexts. The eight mismatches not detected were either within 50 bp of the nearest end of the PCR product or in isolated high-melting-temperature domains. Therefore, it was possible to predict in advance the end regions and sequence contexts in which mismatches may be difficult to detect. The procedure can be applied to any PCR products of 200-800 bp and requires no special equipment or preparation of samples.
700 citations
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TL;DR: It is found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias.
Abstract: In this study, we examined yeast proteins by two-dimensional (2D) gel electrophoresis and gathered quantitative information from about 1,400 spots. We found that there is an enormous range of protein abundance and, for identified spots, a good correlation between protein abundance, mRNA abundance, and codon bias. For each molecule of well-translated mRNA, there were about 4,000 molecules of protein. The relative abundance of proteins was measured in glucose and ethanol media. Protein turnover was examined and found to be insignificant for abundant proteins. Some phosphoproteins were identified. The behavior of proteins in differential centrifugation experiments was examined. Such experiments with 2D gels can give a global view of the yeast proteome.
696 citations
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TL;DR: In this paper, human transforming growth factor beta 1 (TGF-beta 1) was purified as a latent high Mr complex from human platelets by a six-step procedure, and the complex was composed of at least three components with apparent Mr values of 13,000, 40,000 and 125,000-160,000.
691 citations
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TL;DR: A high-resolution method for two-dimensional separation of membrane proteins is described, a modification of the one recently described by O'Farrell (1975); about 150 different proteins can be visualized in Escherichia coli or Salmonella typhimurim cell envelopes.
Abstract: A high-resolution method for two-dimensional separation of membrane proteins is described. It involves a nondiscriminating solubilization of a membrane preparation with sodium dodecyl sulfate, followed by electrophoresis in the first dimension according to charge (by isoelectric focusing). The electrophoresis in the second dimension is in the presence of sodium dodecyl sulfate, thus separating proteins on the basis of molecular weight. Electrophoresis in the first dimension is either on a thin slab gel, or on a small-diameter tube; electrophoresis in the second dimension is on a thin slab gel. Up to 100 mug of protein can be analyzed. The two-dimensional system is a modification of the one recently described by O'Farrell (1975). About 150 different proteins can be visualized in Escherichia coli or Salmonella typhimurim cell envelopes; examples of differences between mutant and wild-type strains are presented. The method is applicable also to membrane preparations from other sources: a two-dimensional separation of plasma membrane proteins from HeLa cells is presented.
690 citations
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TL;DR: With the possible exception of chromosomes that differ greatly in size or electrophoretic behavior from all the known chromosomes, the results appear to define a complete "electrophoretics karyotype" for yeast.
Abstract: The chromosomal DNA molecules of a standard laboratory strain of Saccharomyces cerevisiae have been separated into 12 well-resolved bands by orthogonal-field-alternation gel electrophoresis. DNA X DNA hybridization probes derived from cloned genes have been used to correlate this banding pattern with yeast's genetically defined chromosomes. The 12 bands are shown to represent 9 singlets and 3 comigrating doublets, thereby accounting for 15 chromosomes that were identified as I-XI and XIII-XVI. Because the three comigrating doublets could be readily resolved in certain laboratory yeast strains that contain chromosome-length polymorphisms relative to our standard strain, all 15 of these chromosomes could be displayed as a single band in at least one of four strains that were studied. A 16th chromosome (number XII), which is known to contain the genes for rRNA, does not reproducibly enter the gels. By making use of the band identifications, the previously unmapped fragment F8 was assigned to chromosome XIII. With the possible exception of chromosomes that differ greatly in size or electrophoretic behavior from all the known chromosomes, the results appear to define a complete "electrophoretic karyotype" for yeast.
689 citations