title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey 17033.
authors.forename: M G
authors.initials: MG
authors.lastname: Fried
authors.forename: G
authors.initials: G
authors.lastname: Liu
mesh_terms: D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D007763:Lac Operon / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012995:Solubility
chemical_list: D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial