Electrophoretic assay for DNA-binding proteins

Abstract

This chapter describes a generally applicable assay for specific DNA-binding proteins in crude extracts. The assay is based on gel electrophoretic separation of protein–DNA complexes from each other and from free DNA. This assay employed in several laboratories to detect a variety of DNA-binding proteins. In spite of relatively brief experience with the electrophoretic assay as a detection tool, its exceptionally high sensitivity has already made this assay the method of choice in many experimental settings. The electrophoretic assay has been successfully applied to the detection of both highly sequence-specific, nonabundant DNA-binding proteins and abundant DNA-binding proteins of relatively low nucleotide sequence specificity. After briefly describing some of the alternative techniques suitable for the detection of DNA-binding proteins in crude extracts, it discusses the electrophoretic assay, its recent modifications, and consider other applications of the assay. Although nondenaturing electrophoretic systems have existed for almost as long as the method of gel electrophoresis itself, the arrival of powerful analytical applications of this technology is quite recent and other useful variations of the theme are still to come.