Fluorescent labeling of cysteinyl residues to facilitate electrophoretic isolation of proteins suitable for amino-terminal sequence analysis.
A protein labeling procedure which enables detection of subpicomole quantities of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gels is described. Proteins are rendered fluorescent by reduction of disulfide bonds with dithiothreitol followed by alkylation with 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (5-I-AEDANS) or 5-iodoacetamido-fluorescein. Labeling is performed prior to electrophoresis, thus eliminating the need for staining with dyes and destaining after electrophoresis. As little as 375 fmol (25 ng) of prelabeled bovine serum albumin can be readily visualized after electrophoresis. Bands are still visible after electrophoretic transfer to nitrocellulose. Simultaneous labeling of proteins in complex mixtures is possible using this technique. This includes cysteine containing proteins of disrupted Newcastle disease virus. The magnitudes of the molecular weight increases which occur upon labeling reflect the cysteine contents of proteins. The mode of chemical modification for the prelabeling procedure was chosen because of its compatibility with analytical techniques, such as amino acid analysis, peptide mapping, or sequence analysis, which may be applied to the protein after electroelution from SDS-acrylamide gels. It replaces the need for reduction and carboxymethylation prior to these analytical procedures. Protein-sequence analysis of prelabeled bovine serum albumin, including samples electroeluted from SDS-acrylamide gels, has justified the choice of this method to facilitate isolation of proteins for sequence analysis. Equivalent sequence data were obtained with reduced bovine serum albumin S-alkylated with iodoacetic acid or 5-I-AEDANS.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D005452:Fluoresceins; D005453:Fluorescence; D009282:Naphthalenesulfonates; D011506:Proteins / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate
D005452:Fluoresceins; D009282:Naphthalenesulfonates; D011506:Proteins; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; C015296:5-iodoacetamidofluorescein; C006421:1,5-I-AEDANS; D003545:Cysteine