Sequestration StabilizeslacRepressor–DNA Complexes during Gel Electrophoresis

Abstract

The gel electrophoresis mobility shift assay is widely used for both qualitative and quantitative characterization of protein–nucleic acid interactions. Often it is found that protein–nucleic acid complexes persist within gels for much longer than would be expected on the basis of their free solution lifetimes. Excluded volume and matrix-interaction mechanisms have been proposed to account for the enhanced stabilities of complexes within gels. To test these mechanisms, we have investigated the influences of gel composition and concentration on the pseudo first-order dissociation kinetics of complexes containing theEscherichia colilactose (lac) repressor protein and lactose promoter DNA. In both polyacrylamide and agarose gels, dissociation rates were slower than those in free solution and decreased with increasing gel concentration. This result is inconsistent with mechanisms of stabilization that require specific interactions with the gel matrix. Under standard reaction conditions, free solution values ofkdisswere proportional to [DNA]0.83±0.11, while in 10% polyacrylamide gelskdissvalues were proportional to [DNA]0.48±0.09. These results suggest that the lifetimes oflacrepressor–DNA complexes in free solution are limited by their encounter frequency with molecules of DNA or with protein–DNA complexes; some or all of the stabilization observed in gels may be due to a reduction in this frequency.