Internal amino acid sequence analysis of proteins after limited proteolytic cleavage

A procedure was used to obtain peptide fragments for sequence analysis from proteins separated by gel electrophoresis. After separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were briefly stained with Coomassie blue, interested protein-containing bands were cut and loaded onto the wells of a new SDS-PAGE gel. The protein was digested by protease V8 during electrophoresis, the resulting peptide fragments were electroblotted onto polyvinylidene difluoride membrane, and then sequenced with protein sequencer. The method can be used not only to obtain amino acid sequences from N-terminal blocked proteins, but also to produce multiple and independent amino acid sequence information from normal proteins.