'In gel' cleavage with cyanogen bromide for protein internal sequencing.
J Biochem Biophys Methods
; 35(1): 1-10, 1997 Aug 01.
Article
em En
| MEDLINE
| ID: mdl-9310863
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.
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01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas
/
Análise de Sequência
/
Eletroforese em Gel de Poliacrilamida
Idioma:
En
Ano de publicação:
1997
Tipo de documento:
Article