INTERNAL SEQUENCES FROM PROTEINS DIGESTED IN POLYACRYLAMIDE GELS

A simple method for proteolytic digestion of Coomassie blue-stained pr oteins in a polyacrylamide matrix is presented. It consists of first r educing and alkylating the stained proteins with dithiothreitol and io doacetamide in the presence of 0.1% sodium dodecyl sulfate and subsequ ent digestion with the endoproteinase LysC. The reduction and alkylati on step was introduced since experiments with lysozyme and ribonucleas e A showed that extremely complex peptide patterns were obtained if no precautions were taken to suppress disulfide bond formation during in -gel digestion of proteins. The advantage of this method is that no bl otting step is required for generating internal sequences and that ext ensive proteolysis occurs which closely resembles that resulting from solution digests. The method has been successfully used to generate in ternal sequence data from low microgram quantities of proteins excised from a-dimensional Coomassie blue-stained gels. (C) 1995 Academic Pre ss, Inc.