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Proteome analysis of polyacrylamide gel‐separated proteins visualized by reversible negative staining using imidazole‐zinc salts
Author(s) -
CastellanosSerra Lila,
Proenza Wilfredo,
Huerta Vivian,
Moritz Robert L.,
Simpson Richard J.
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19990101)20:4/5<732::aid-elps732>3.0.co;2-q
Subject(s) - chromatography , chemistry , polyacrylamide gel electrophoresis , bottom up proteomics , proteome , proteolysis , silver stain , polyacrylamide , capillary electrophoresis , gel electrophoresis , proteases , mass spectrometry , protease , sample preparation , tandem mass spectrometry , biochemistry , protein mass spectrometry , biology , microbiology and biotechnology , enzyme , polymer chemistry
Identification and characterization of proteins isolated from natural sources by polyacrylamide gel electrophoresis has become a routine technique. However, efficient sample proteolysis and subsequent peptide extraction is still problematic. Here, we present an improved protocol for the rapid detection of polyacrylamide gel‐separated proteins, in situ protein modification, proteolytic digestion and peptide extraction for subsequent protein identification and characterization by capillary high‐performance liquid chromatography/tandem mass spectrometry. This simple technique employs the rapid imidazole‐zinc reverse stain, in‐gel S‐pyridylethylation and proteolytic digestion of microcrushed polyacrylamide gel pieces with proteases. This technique obviates the need for buffer exchange or gel lyophilisation due to all of the sample manipulation steps being carried out at near neutral pH and thus lends itself readily to automation.