An EMSA-based method for determining the molecular weight of a protein--DNA complex.

We have developed an electrophoretic method to determine the size of a protein—DNA complex using the same binding and electrophoresis conditions as in an electrophoretic mobility shift assay (EMSA). The method is an adaptation of that of Ferguson (1), originally developed to determine the size of native proteins in non-denaturing gel systems. In SDS polyacrylamide gel electrophoresis, association of SDS gives all proteins equal charge per unit mass and also helps to unfold the protein. Thus the major determinant of protein mobility in an SDS gel is the length of the polypeptide chain. In contrast, in non-denaturing gels, mobility is influenced by molecular weight, conformation and charge. Ferguson analysis allows the molecular weight of a native protein to be determined indirectly by electrophoresing it, along with a number of standard proteins, on a series of non-denaturing tube gels of different polyacrylamide concentrations. Since the ionic conditions are identical in each experiment, the decrease in mobility observed as the polyacrylamide concentration is increased is due entirely to the sieving effects of the gel and hence is related to the species' size and shape but not its charge. The molecular weight of the unknown can be determined graphically from this change in mobility, as detailed below.