DETERMINATION OF THE MOLECULAR-WEIGHT OF DNA-BOUND PROTEIN(S) RESPONSIBLE FOR GEL-ELECTROPHORETIC MOBILITY SHIFT OF LINEAR DNA FRAGMENTS EXEMPLIFIED WITH PURIFIED VIRAL MYB PROTEIN

A protein-DNA complex has less gel electrophoretic mobility than the free DNA fragment. One parameter for the degree of retardation of a linear DNA fragment in a protein-DNA complex is the molecular weight of the bound protein(s). The quotient of the migration distances of free DNA (m) and protein-DNA complex (m'') is a function of the molecular weight (MW) of the bound protein(s). Based on the evaluation of the lac repressor induced mobility shift of a 203 bp DNA fragment containing the lac operator in a 50% nondenaturing polyacrylamide gel a direct proportionality could be shown between (m/m''-1) and MW with the proportionality factor K = 215 kDa. The factor K depends on the acrylamide concentration in the gel, getting lower values with increasing acrylamide concentrations. A calculation is given to determine the molecular weight of DNA-binding factors responsible for the decreased electrophoretic mobility of a linear DNA fragment. As an example this calculation was used in order to analyse DNA-binding of the isolated viral mvb protein. It could be demonstrated that the viral mvb protein binds to DNA as a monomer and a dimer.