Molecular sequestration stabilizes CAP-DNA complexes during polyacrylamide gel electrophoresis.

Abstract

The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes that are short-lived in free solution (t1/2 of the order of minutes) persist for hours under the conditions of gel electrophoresis. We have investigated the influence of polyacrylamide gels on the pseudo first-order dissociation kinetics of complexes containing the E.coli cyclic AMP receptor protein (CAP) and lactose promoter DNA. Within the gel matrix, kdiss decreased with increasing [polyacrylamide] and the order of the reaction was changed. In free solution, kdiss was proportional to [DNA]2, while in 5% gels, kdiss was proportional to [DNA]0.3. In gels of [polyacrylamide] > or = 10%, kdiss was nearly independent of [DNA] until fragment concentrations exceeded 0.1 microM. Even in the absence of competing DNA, kdiss(gel) < kdiss(solution). These results suggest that the lifetime of CAP-DNA complexes in free solution is 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.