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 using a slot-blot apparatus, incubating with 32P-labeled DNA probe in buffer in the presence of excess of nonspecific E. coli DNA at room temperature, and washing with increasing concentration of NaCl (from 50 to 500 mM) to obtain optimum signal. A simple and rapid scheme of purification of a sex and tissue-specific DNA-binding protein, which binds specifically to the GATA repeats of Bkm (banded krait minor satellite DNA), 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) containing 66 copies of GATA repeats was used to prepare the sequence-specific 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 in general.
Majumdar
K C
KC
Centre for Cellular and Molecular Biology, Hyderabad, India.
Shetty
S
S
Wadhwa
R
R
Bhaskar
S
S
Ganesan
M
M
Singh
L
L
D000818:Animals; D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D004330:Drosophila; D004926:Escherichia coli / Q000235:genetics; D015336:Molecular Probe Techniques; D017421:Sequence Analysis
D015342:DNA Probes; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins