IN GEL CLEAVAGE WITH CYANOGEN-BROMIDE FOR PROTEIN INTERNAL SEQUENCING

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) i s a powerful purification technique in protein chemistry research. Thi s procedure is frequently used as a last step in protein purification for sequencing. For proteins which are N-terminal blocked, 'in gel' di gestion offers a useful approach for the generation of internal sequen ce data from proteins purified by SDS-PAGE. In this study, we Propose a procedure where proteins purified by this method are chemically clea ved 'in gel' by using CNBr and the resulting peptides are isolated in a second SDS-PAGE. After that, electroblotting is performed onto PVDF membranes and the electroblotted and Coomassie-stained peptide from ea ch band is then sequenced by Edman degradation. Proteins often have a small number of methionines whose cleavage allows the obtention of lon g peptides suitable to sequence a good deal of residues. Three standar d proteins of different molecular mass have been assayed by this proce dure and the 'in situ' cleavage profile compared with direct chemical digestion in a protein solution. (C) 1997 Elsevier Science B.V.