Stabilizing Labile DNA-Protein Complexes in Polyacrylamide Gels

Abstract

The electrophoretic mobility shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA-protein interactions. The technique uses polyacrylamide gel electrophoresis to separate DNA-protein or RNA-protein complexes from free DNA or RNA. Polyacrylamide gels stabilize DNA-protein, RNA-protein, or protein-protein complexes by a crowding or caging mechanism. Still every technique has its limitations. EMSA, as standardly practiced today, works well for complexes with association binding constants Ka>109 M−1 under normal conditions of salt and pH. Many DNA-protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. We take advantage of our previous observation that neutral osmolytes can strongly slow down the rate of DNA-protein complex dissociation to develop a method that uses osmotic stress to stabilize complexes in the gel matrix as well as in the solution. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using the electrophoretic mobility shift assay. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein-DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and capillary electrophoresis is straightforward.