Filter binding assays for topoisomerase-DNA complexes

Abstract

Protein-nucleic acid interactions have been studied by density shift gradients, affinity columns, and gel retardation. Some of these methods are tedious or have severe limitations in quantitative analysis. Filter binding assays are fast, sensitive, and suitable for physical chemical measurements (1,2). Filter binding techniques are based on the observation that proteins, either free or complexed with nuclei acids, bind quantitatively to certain materials in conditions that free RNA and DNA are not retained. Experimental data can thus be obtained from analysis of the nucleic acids retained in the filter, or found in filtrates and washes. Because protein-adsorbing materials can differ in their efficiency, capacity, and flow characteristics, filters from the same lot should be used in serial experiments, and the optimal filtration conditions need to be empirically determined in each case