S-carboxymethylation of proteins transferred onto polyvinylidene difluoride membranes followed by in situ protease digestion and amino acid microsequencing.
S-Carboxymethylation of immobilized proteins, separated by gel electrophoresis and transblotted to polyvinylidene difluoride (PVDF) membranes is described. In situ protease digestion was used to produce peptide fragments in high yields that were suitable for microsequencing. These fragments could also be used to produce peptide maps, obtained by reverse-phase high performance liquid chromatography (HPLC), that exhibited excellent reproducibility and were comparable to results obtained from digestions in solution. This technique makes it possible to identify Cys residues during Edman degradation. Approximately 70% of the amino acid residues from a protein were determined using only 50-200 pmol. Sufficient information for production of oligonucleotide probes was obtained using 10-50 pmol of material applied to a polyacrylamide gel.
Iwamatsu
A
A
Central Laboratories for Key Technology, Kirin Brewery, Gunma, Japan.
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D005069:Evaluation Studies as Topic; D008567:Membranes, Artificial; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D005465:Fluorocarbon Polymers
D010450:Endopeptidases; D008567:Membranes, Artificial; D010446:Peptide Fragments; D011145:Polyvinyls; D011506:Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers