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Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels

Time:2015/11/12 3:05:20

Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: Results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes

An improved method is described for the renaturation of microgram amounts of proteins from sodium dodecyl sulfate-polyacrylamide gels. The protein band is visualized in the gel by KCl staining, the band cut out and crushed, and the protein eluted by diffusion in a buffer containing 0.1% sodium dodecyl sulfate. Protein is concentrated and sodium dodecyl sulfate is removed by acetone precipitation of the sample. Renaturation of the protein occurs after the precipitate is dissolved in 6 m guanidine hydrochloride and then diluted. The activity of the sigma subunit of Escherichia coli RNA polymerase can be recovered with 98–100% efficiency after electrophoresis in an SDS-gel and renaturation by this technique. To assess whether the method is generally applicable we tested some or all of the steps involved in the procedure using E. coli transcription termination factor rho, β-galactosidase, alkaline phosphatase, wheat α-amylase, and DNA topoisomerase. We show how the method can be used to determine the approximate molecular weight of the DNA topoisomerase polypeptide by sectioning a gel on which a partially pure sample has been fractionated by electrophoresis.

Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate

Physical factors leading to the separation of oligopeptides in the molecular weight range of 1,200 to 10,000 daltons by analytical-scale electrophoresis in polyacrylamide gel with sodium dodecyl sulfate are described. Increased acrylamide concentration, cross-linkage, and inclusion of 8 M urea to decrease gel porosity, increased gel length, and buffer ions of low mobility are factors which yield improved separation of such peptides. Electrophoretic mobilities of eleven peptides were linearly related to the logarithm of their molecular weights with a standard deviation of 18% in a system of improved resolution. The intrinsic charge and conformation of peptides were found to be relatively more important determinants of electrophoretic mobilities than for proteins larger than 10,000 daltons. Such determinants were relatively more important with four of the eleven peptides examined, leading to deviations from the log-linear slope greater than 18%. Because of the importance of intrinsic charge and conformation, the system, although allowing a first approximation in molecular weight determination, may also be applicable to peptide “mapping,” particularly for “insoluble” peptide mixtures with prominent hydrophobic association, such as encountered in cellular membranes, viruses, and proteolytic digests.