Sample preparation by SDS/PAGE and in-gel digestion.

Abstract

A convenient method for the isolation of samples for microsequencing or mass spectrometry utilizing polyacrylamide gel electrophoresis is described in detail, including pre-requisites for a successful outcome. Briefly, after visualization by e.g. Coomassie or silver, internal peptides are generated by in-gel proteolytic digestion, and after extraction, the corresponding protein is analyzed, i.e. the protein is identified by search in sequence data banks using a peptide sequence or a peptide mass map. Alternatively, a novel protein is detected, and an oligonucleotide probe for subsequent molecular biology work is synthesized, based on the obtained peptide sequence. One obvious advantage is that the procedure allows practically any protein to be studied. In-gel digestion has become the fundamental means of preparing samples in the many ongoing proteome projects. A few representative examples are given.