Reassessment of commercially available molecular weight standards for peptide sodium dodecyl sulfate-polyacrylamide gel electrophoresis using electroblotting and microsequencing

Myoglobin CNBr peptides, constituting the commercially available molecular weight calibration kits for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were analyzed by microsequencing after electroblotting on polyvinylidene difluoride (Immobilon) membranes. An obvious disagreement was found between peptide identification and the data provided by the manufacturers. We observed 6 peptides from Mr 2500 to 17,000 corresponding, in increasing size order, to the 3 peptides resulting from the total CNBr digestion, to 2 incompletely cleaved peptides and to the intact myoglobin. Using a corrected calibration curve, a linear relationship was established from Mr 6000 to 43,000 and a second one for shorter peptides. This method of electrophoresis and electroblotting, easily adapted for peptides, is a powerful tool for peptide identification correlated with size determination. It is especially useful for CNBr-cleaved peptides.