A blotting procedure which preserves specific protein-DNA interactions.
A quick, quantitative, and nonselective electrophoretic transfer of proteins from acetic acid-urea gels onto nitrocellulose, which preserves their ability to interact specifically with DNA, is achieved when exposure to dodecyl sulfate ions is avoided and a special type of nitrocellulose is used which contains cellulose phosphate ester. Filter-adsorbed histone H1 and other nuclear proteins from an insect, Chironomus thummi, were tested for binding of an AT-rich DNA sequence from the heterochromatin of the same organism under competitive conditions. On the blots, histone H1 exhibited the dependency of DNA binding on NaCl concentration and the preference for AT-rich DNA or poly[d(A-T)] found in quantitative filter-binding studies. By stepwise alteration of the NaCl molarity and competing Escherichia coli DNA concentration, respectively, in the binding buffer, two minor protein fractions could be identified in the heterogeneous extracts, one of which bound preferentially to AT-rich DNA, and the other bound to this sequence at up to 500 mM NaCl. Exposure to dodecyl sulfate led to a disappearance of the ability of these proteins to interact specifically with DNA. While nondenaturing transfer by diffusion (L. Levinger and A. Varshavsky (1982) Proc. Natl. Acad. Sci. USA 79, 7152) is a procedure that requires about 2 days, the present technique of gentle protein transfer for DNA binding studies requires only 2 to 3 h.
Wendler
I
I
III. Zoologisches Institut-Entwicklungsbiologie, Universität Göttingen, Federal Republic of Germany.
Grossbach
U
U
D000818:Animals; D002683:Chironomidae / Q000032:analysis; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting / Q000379:methods; D007124:Immunoenzyme Techniques; D007814:Larva / Q000032:analysis
D015342:DNA Probes; D004268:DNA-Binding Proteins