Molecular sequestration stabilizes CAP-DNA complexes during polyacrylamide gel electrophoresis

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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 (tfrac; 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, 'fdiss was proportional to [DNA]0.3, while in 5% gels, kdiss was proportional to [DNA]diss. In gels of [polyacryl-amide] ≥ 10%, kdiss was nearly independent of [DNA] until fragment concentrations exceeded 0.1 μM. 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.

Original languageEnglish
Pages (from-to)5054-5059
Number of pages6
JournalNucleic Acids Research
Issue number23
StatePublished - Nov 25 1994

Bibliographical note

Funding Information:
This work was supported by National Science Foundation grant DMB-91-48816

ASJC Scopus subject areas

  • Genetics


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