Calibrating Protein Molecular Weight

The same molecular weight standard may have slightly different mobility, resulting in different apparent molecular weight when run in different SDS-PAGE buffer systems. If you are using the Novex® protein molecular weight standards, use the apparent molecular masses of these standards in the gels listed in Tables 5 through 8 to determine an apparent molecular weight of your protein.

Apparent molecular masses of Mark 12™ Unstained Standards and Novex® Sharp Prestained Protein Standards under various gel and buffer conditions

Apparent molecular masses of SeeBlue® and SeeBlue® Plus2 Prestained Protein Standards under various gel and buffer conditions

Apparent molecular masses of Novex® Sharp Prestained Protein Standards and Mark 12™ Unstained Standards on Tris-glycine and Tricine gels

Apparent molecular masses of SeeBlue® and SeeBlue® Plus2 Prestained Protein Standards on Tris-glycine and Tricine gels

When using SDS-PAGE for molecular weight determination, slight deviations from the calculated molecular weight of a protein (calculated from the known amino acid sequence) can occur due to the retention of varying degrees of secondary structure in the protein, even in the presence of SDS. This phenomenon is observed in highly organized secondary structures (collagens, histones, or highly hydrophobic membrane proteins) and in peptides, where the effect of local secondary structure becomes magnified relative to the total size of the peptide.4 kDa

Slight differences in protein mobilities also occur when the same proteins are run in different SDS-PAGE buffer systems. Each SDS-PAGE buffer system has a different pH, which affects the charge of a protein and its binding capacity for SDS. The degree of change in protein mobility is usually small in natural proteins but more pronounced with “atypical” or chemically modified proteins, such as prestained standards.