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Staining of sodium dodecyl sulfate gels

Time:2015/11/12 3:08:01

Elution of proteins from SDS-polyacrylamide gels, removal of SDS, and renaturation of enzymatic activity: results with sigma subunit of E. 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. 

Elution of proteins from SDS-polyacrylamide gels, removal of SDS, and renaturation of enzymatic activity: results with sigma subunit of E. coli RNA polymerase, wheat germ DNA topoisomerase and other enzymes - ResearchGate. Available from: http://www.researchgate.net/publication/222223283_Elution_of_proteins_from_SDS-polyacrylamide_gels_removal_of_SDS_and_renaturation_of_enzymatic_activity_results_with_sigma_subunit_of_E._coli_RNA_polymerase_wheat_germ_DNA_topoisomerase_and_other_enzymes [accessed Nov 12, 2015].

Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels.

A new modification of silver staining of proteins in sodium dodecyl sulfate polyacrylamide gels is adapted to automated staining in PhastSystem Development Unit. The use of a reduction step, after fixation, with thiosulfate in alcoholic sodium acetate buffer results in a considerable increase in sensitivity without the need for a recycling step. The detection limit is tenfold lower than in the silver staining procedure recommended so far for PhastSystem and corresponds to 0.05-0.1 ng protein per band. Total staining time with the new procedure is 75 min.