Informace o publikaci
Evaluation of dissociation constants from competition binding experiments based on the relative binding ratio
| Autoři | |
|---|---|
| Rok publikování | 2011 |
| Druh | Článek v odborném periodiku |
| Časopis / Zdroj | Analytical Biochemistry |
| Fakulta / Pracoviště MU | |
| Citace | |
| Doi | http://dx.doi.org/10.1016/j.ab.2010.09.023 |
| Obor | Analytická chemie, separace |
| Klíčová slova | Protein–DNA interactions Electrophoretic mobility shift assay Competition binding experiment |
| Popis | 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 selfcompetition 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. |