Digestion of Proteins in Gels for Sequence Analysis

Abstract

AbstractA high percentage of eukaroytic proteins have blocked amino termini, so it is usually necessary to cleave the protein chemically or enzymatically to obtain the partial sequences needed for cDNA cloning. Because SDS‐PAGE is the current method of choice for the final purification of the >25 pmol amounts of protein that are usually required for internal sequencing, procedures that can be used to digest proteins in situ in SDS‐polyacrylamide gels are often the most useful and have the added benefit of eliminating losses that may occur during blotting. Two strategies have been developed to respond to this need and to deal with the unique problems posed by SDS, which are that SDS inhibits trypsin, one of the enzymes that is most commonly used for internal sequencing studies, and also interferes with reversed‐phase HPLC. In this unit, SDS is removed from the gel prior to enzymatic cleavage by the staining and subsequent washing steps. An acetonitrile wash is performed to remove residual SDS from the protein sample, and Tween 20 is then added back to the sample to maintain the solubility of the denatured protein. In an alternate protocol, the gel slices are washed with ammonium bicarbonate and the protein samples digested in the absence of detergent. Support protocols are provided for determining the amount of protein present in the sample via amino acid analysis, and for reducing and alkylating proteins separated by SDS‐PAGE (which facilitates the identification of cysteine residues during subsequent peptide sequencing reactions).