Determination of Molecular Mass by SDS-PAGE

The independent determination of molecular masses by SDS-PAGE is impossible. To estimate the molecular mass of a protein, measure the path of that protein or calculate its Rf value (distance of the protein from origin/distance of electrophoresis front from origin) and compare these values with that of marker proteins, i.e., proteins with independently determined molecular masses. This method is successful only if the protein of interest behaves regularly in SDS-PAGE, i.e., it is totally unfolded by SDS, has a rod-like shape, and the SDS/protein ratio is the same for the unknown and the marker protein. Especially highly hydrophobic proteins and glycoproteins often deviate from these assumptions. 

Calculation of the molecular mass of an unknown protein follows the same procedure as, for example, quantitative protein determination plotting of the Rf of the calibration proteins against their molecular mass computing of a standard curve and estimation of the MW of the unknown protein and using the regression functions of the standard curve (c.f Fig. 2.1). 

Hames BD(1990) In Hames BD, Rickwood D (eds.) Gel electrophoresis of proteins a practical approach, 2nd ed. IRL Press, Oxford, p 16 

---