title: 'In gel' cleavage with cyanogen bromide for protein internal sequencing.
abstract: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a powerful purification technique in protein chemistry research. This procedure is frequently used as a last step in protein purification for sequencing. For proteins which are N-terminal blocked, 'in gel' digestion offers a useful approach for the generation of internal sequence data from proteins purified by SDS-PAGE. In this study, we propose a procedure where proteins purified by this method are chemically cleaved '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 each band is then sequenced by Edman degradation. Proteins often have a small number of methionines whose cleavage allows the obtention of long peptides suitable to sequence a good deal of residues. Three standard proteins of different molecular mass have been assayed by this procedure and the 'in situ' cleavage profile compared with direct chemical digestion in a protein solution.
authors.affiliation: Laboratorio Nacional de Investigación, Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
authors.forename: O L
authors.initials: OL
authors.lastname: Córdoba
authors.forename: S B
authors.initials: SB
authors.lastname: Linskens
authors.forename: E
authors.initials: E
authors.lastname: Dacci
authors.forename: J A
authors.initials: JA
authors.lastname: Santomé
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D017421:Sequence Analysis / Q000379:methods*; D012967:Sodium Dodecyl Sulfate
chemical_list: D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D003488:Cyanogen Bromide