Mapping of radiolabeled peptides derived from proteolysis of polypeptides bound to nitrocellulose after "Western" blotting.
Sections of nitrocellulose containing bound 32P-labeled polypeptides were excised from "Western" blots and exhaustively digested by trypsin in order to analyze the distribution of phosphorylation sites between the products of limited proteolysis of the multifunctional protein CAD. Using the criterion of analytical isoelectric focusing, the 32P-peptides obtained by this method were found to be similar, although not identical, to peptides obtained by a more conventional digestion of trichloroacetic acid precipitates. Digestion on Western blots is more straightforward than electrophoretic elution of individual gel slices, gives better recoveries than direct digestion of gel slices, and is particularly suitable for peptide mapping of small peptides which bind to nitrocellulose but would diffuse out of polyacrylamide gels during the commonly used fixing and staining procedures.
D001221:Aspartate Carbamoyltransferase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D003101:Collodion; D004080:Dihydroorotase; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007526:Isoelectric Point; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis*; D010447:Peptide Hydrolases / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis*
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D003101:Collodion; D001221:Aspartate Carbamoyltransferase; D010447:Peptide Hydrolases; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)