Internal sequences from proteins digested in polyacrylamide gels.
A simple method for proteolytic digestion of Coomassie blue-stained proteins in a polyacrylamide matrix is presented. It consists of first reducing and alkylating the stained proteins with dithiothreitol and iodoacetamide in the presence of 0.1% sodium dodecyl sulfate and subsequent digestion with the endoproteinase LysC. The reduction and alkylation step was introduced since experiments with lysozyme and ribonuclease 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 blotting step is required for generating internal sequences and that extensive proteolysis occurs which closely resembles that resulting from solution digests. The method has been successfully used to generate internal sequence data from low microgram quantities of proteins excised from 2-dimensional Coomassie blue-stained gels.
Jenö
P
P
Department of Biochemistry, Biozentrum of the University of Basel, Switzerland.
Mini
T
T
Moes
S
S
Hintermann
E
E
Horst
M
M
D000180:Acrylic Resins; D000478:Alkylation; D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping
D000180:Acrylic Resins; C016680:polyacrylamide gels