A new affinity purification procedure for DNA-binding proteins using bromoacetyl agarose

摘要

Characterization of proteins binding to the promoters of eukaryo-tic genes has proved essential for understanding the transcrip-tional regulation of viral and cellular genes. Because of the low abundance of these proteins in the cell, conventional purification of these molecules has been laborious. In contrast, sequence-specific DNA affinity chromatography has greatly faciliated rapid isolation and characterization of DNA-binding proteins. A number of procedures for coupling specific DNA sequences toresins have been reported to be effective (1-5). However, all have restrictions on the form of DNA which can be attached to the matrix. In case of avidin or streptavidin columns, biotin must be coupled to the DNA (1,2). Cyanogen bromide activated coupling requires single-stranded overhangs (3). A more recent technique by Larson and Verdine allows efficient coupling of blunt-ended double-stranded ODNs, but requires the use of complex modified nucleosides during DNA synthesis (4).