DETECTION AND PURIFICATION OF SEQUENCE-SPECIFIC DNA-BINDING PROTEIN

A simple assay for identifying DNA-binding proteins is described that involves loading of protein fractions onto nitrocellulose membrane usi ng a slot-blot apparatus, incubating with P-32-labeled DNA probe in bu ffer in the presence of excess of nonspecific E. coli DNA at room temp erature, and washing with increasing concentration of NaCl (from 50 to 500 mM) to obtain optimum signal, A simple and rapid scheme of purifi cation of a sex and tissue-specific DNA-binding protein, which binds s pecifically to the GATA repeats of Bkm (banded krait minor satellite D NA), designated as Bkm-binding protein (BBP), is also described. This requires only a DNA affinity column after the initial ammonium sulfate precipitation. The insert (545 bp) of the Drosophila clone 2(8) conta ining 66 copies of GATA repeats was used to prepare the sequence-speci fic DNA-Sepharose affinity column. The slot-blot-binding assay and the simple scheme of purification described here may be used for routine screening and purification of sequence-specific DNA-binding proteins i n general. (C) 1996 Academic Press, Inc.