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.