Molecular sequestration stabilizes CAP-DNA complexes during polyacrylamide gel electrophoresis. |
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Authors: | M G Fried and G Liu |
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Institution: | Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey 17033. |
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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. |
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