Gas-phase sequencing after electroblotting on polyvinylidene difluoride membranes assigns correct molecular weights to myoglobin molecular weight markers

Abstract Commercially available polypeptide marker kits containing peptides generated by cyanogen bromide cleavage of either horse heart myoglobin or sperm whale myoglobin have been investigated by sodium dodecyl sulfate — polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting on polyvinylidene difluoride membranes, and gas-phase sequencing. It could be shown that the molecular weights assigned to the SDS-PAGE bands by the companies are incorrect. Arranged in descending order, the marker kits are composed of the following polypeptide fragments from myoglobin: positions 1–153, 1–131, 56–153, 56–131, 1–55, and 132–153. A polypeptide comprising residues 1–14 was not found. According to these results the log M r versus R f plot used for calibration must be revised. For the separation of low molecular weight polypeptides and peptides a new gel system based on the theory of multiphasic zone electrophoresis combined with a modified Coomassie staining procedure is reported.