Isolation of proteins for peptide mapping, amino acid analyses, and sequencing using disulfide crosslinked polyacrylamide gels.
Conditions for recovery of small amounts of proteins (1-50 micrograms) from disulfide crosslinked polyacrylamide gels have been examined. Procedures were developed for solubilization and precipitation of Coomassie blue-stained protein bands excised from gels after electrophoretic separations. The precipitated protein was then resolubilized for use in peptide mapping, amino acid analyses, or microsequencing. The amino acid compositions of standard proteins (bovine albumin, ovalbumin, phosphorylase b, and beta-galactosidase) isolated by this method were in good agreement with the values for the corresponding conventionally purified proteins. Sequencing was done with high repetitive yield on samples of 100 pmol or below. The method has been successfully applied to several proteins and protein fragments.
Ghaffari
S H
SH
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Dumbar
T S
TS
Wallace
M O
MO
Olson
M O
MO
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D010449:Peptide Mapping; D011506:Proteins / Q000302:isolation & purification*; D012996:Solutions
D000596:Amino Acids; D003432:Cross-Linking Reagents; D004220:Disulfides; D011506:Proteins; D012996:Solutions