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

AUTOR(ES)

Fried, M G

RESUMO

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.

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