Filter-Binding Assays

Abstract

Membrane filtration has a long history in the analysis of proteinnucleic acid complex formation, having first been used to examine RNA-protein interactions (1), before being introduced to DNA-protein interaction studies by Jones and Berg in 1966 (2). The principle of the technique is straightforward. Under a wide range of buffer conditions, protein-free nucleic acids pass freely through membrane filters, whereas proteins and their bound ligands are retained. Thus, if a particular protein binds to a specific DNA sequence, passage through the filter will result in retention of a fraction of the protein-DNA complex by virtue of the protein component of the complex. The amount of DNA retained can be determined by using radioactively labeled DNA to form the complex and then determining the amount of radioactivity retained on the filter by scintillation counting. The technique can be used to analyze both binding equilibria and kinetic behavior, and, if the DNA samples retained on the filter and in the filtrate are recovered for further processing, the details of the specific binding site can be probed by interference techniques.