Expeditious Molecular Weight Determination of Native Proteins by Transverse Pore Gradient Polyacrylamide Gel Electrophoresis Using the PhastGel System

Abstract

The Ferguson plot is well established method for the molecular weight determination of native proteins and protein complexes. The Ferguson plot requires running three or more gels of different polyacrylamide concentrations to determine the relationship between band migration and polyacrylamide gel concentration and using it solve for an unknown. Herein, we describe a new approach to a Ferguson plot that requires only one 8-25% gradient polyacrylamide gel, run as a transverse pore gradient, to provide the informational equivalent of 10-12 single concentration polyacrylamide gels. Using Cytochrome-C (14.4 kDa), E-GST (25 kDa), BSA (66 kDa) and BSA Dimer (132 kDa) as standards, we are able to determine the molecular weight of E-GST dimer with 9% experimental error in under 3 hours. This method has a significant advantage in reproducibility, efficiency, and cost effectiveness as compared to the standard method used for creating a Ferguson plot and is readily useable by any lab with a PhastSystem (GE LifeSciences).