FACTORS THAT AFFECT THE STABILITY OF PROTEIN-DNA COMPLEXES DURING GEL-ELECTROPHORESIS

The gel electrophoresis mobility shift assay is widely used for qualit ative and quantitative characterization of protein complexes with nucl eic acids. Often it is found that complexes persist within electrophor esis gels for much longer than expected on the basis of their free-sol ution lifetimes. Volume exclusion, direct interaction with gel matrice s and the reduction of water activity by the gel have been proposed as mechanisms enhancing the stability of complexes during electrophoresi s. We have used the well-characterized interaction of the E. coli cycl ic AMP receptor protein (CAP) with lactose promoter DNA to test these proposals. We found that the activity of water within polyacrylamide g els differs little from that of the buffer in which they were cast and that the dependence of the dissociation rate constant on water activi ty is too small for osmotic stabilization to contribute significantly to the lifetimes of CAP-DNA complexes. In addition, we found that a cr oss-linked gel matrix is not required for the stabilization of CAP-DNA complexes, that comparable stabilization is produced by three dissimi lar polymers (linear polyacrylamide, dextran and polyethylene glycol), and that these polymers stabilize complexes more effectively than equ ivalent weight concentrations of their cognate monomers. While these r esults challenge the notion that direct interaction with the gel matri x contributes to the stability of protein-DNA complexes, they are all features expected of excluded volume mechanisms.