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

Abstract

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.