Peptide production from proteins separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis.
The development of amino acid sequencers with subnanomolar sensitivities has increased the need for both selective and highly efficient methods for both protein and peptide isolation. In this paper, we describe a simple procedure that utilizes the high resolving capacity of polyacrylamide gel electrophoresis to isolate a single target polypeptide, which can subsequently be subjected to proteolytic digestion and sequencing. Polypeptides are visualized in polyacrylamide gels as dodecyl sulfate/protein complexes, which are passively diffused from gel slices. Free dodecyl sulfate eluted with the protein solution is removed by KCl precipitation, allowing protein digestion with small amounts of trypsin or other proteolytic enzymes. Following enzymatic digestion, the peptide solution is made 6 M guanidine-HCl to remove interfering contaminants and thereby improve resolution of the digest by reverse-phase high-performance liquid chromatography. The peptides generated by this method are suitable for amino acid sequencing with good overall yields, averaging 15-30% on a gas-phase sequenator. The method described is useful for obtaining multiple peptide sequences from a single polypeptide isolated from a complex protein mixture.
Prussak
C E
CE
Department of Medicine, University of California, San Diego, La Jolla 92093.
Almazan
M T
MT
Tseng
B Y
BY
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D019915:DNA Primase; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007202:Indicators and Reagents; D051379:Mice; D010455:Peptides / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification*; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate
D000596:Amino Acids; D007202:Indicators and Reagents; D010455:Peptides; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases