Evaluation of dissociation constants from competition binding experiments based on the relative binding ratio.
Methods probing protein-DNA associations include direct binding titrations and competition binding experiments. For the latter, we present here a simple procedure allowing the quantitative evaluation of dissociation constants. We show that the ratio between the fraction of a DNA probe bound to protein in the absence of competitor and that in the presence of competitor is, at large competitor concentrations, a linear function of the competitor concentration, and we derive equations allowing the dissociation constant for the protein-competitor complex to be evaluated from the slope. We show further that a self-competition experiment, where the DNA probe and competitor are chemically the same species, can be used as a complement to a direct titration to determine the fraction of protein that is correctly folded for specific DNA binding. Thus, such a combination of direct and self-competition titration can be used as a check of the conformational purity of DNA binding proteins.
Kozelka
Jiří
J
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université Paris Descartes, UMR 8601 CNRS, 75270 Paris, France. kozelka.jiri@gmail.com
D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D007700:Kinetics; D008962:Models, Theoretical; D011485:Protein Binding
D004268:DNA-Binding Proteins; D004247:DNA