Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography

Abstract

DNA-affinity chromatography was first used by Kadonaga and Tjian (1986) for the purification of the transcription factor, Sp1. Oligonucleotides containing the protein-binding site are phosphorylated, ligated and covalently bound to Sepharose. Nuclear proteins are then incubated with the affinity matrix in the presence of non-specific competing DNA, and the protein binding specifically to the oligonucleotides is eluted with high salt. This method has been used widely for the purification of a variety of DNA-binding proteins (e. g. Fletcher et al., 1987; Prywes and Roeder 1987; Bagchi et al., 1987; Sawadogo et al., 1988; Lenardo et al, 1988; Lichtensteiner and Schibler 1989).