DNA binding proteins present in guanidinium isothiocyanate lysates of cells are suitable for specific binding site blotting.

The GITC/CsCl method (1) is frequently utilized for the isolation of intact RNA. Here we describe the use of this method for the extraction of DNA binding proteins suitable for specific binding site blotting. Cells are tysed by guanidinium isothiocyanate and after the overnight ultracentrifugation step the supernatant of the gradient containing denatured cellular proteins is diarysed against buffer A (50% glyceroL, 50 mM NaCL, 10 mM HEPES pH 7.9, 05 mM PMSF, 05 mM DTT). The diarysate is frozen in ahquots at -20*C. For the detection of DNA binding proteins 100 ug of protein prepared by the GITC/CsCl method (described above) or salt-extracted from isolated nuclei (2) are size-fractionated on a SDS-Laemmli-gel and electrotransferred to nitrocellulose overnight at 150 mA in buffer B (1.92 M gryedne, 0.25 M Tris pH 83). The protocol developed by Vinson et aL (3) for binding site screening of protein expression libraries is used for further processing of the protein filters. All following steps are carried out at 4*C. The filter (13xl8cm) is incubated twice for 10 min in 50 ml buffer C (25 mM Nad, 25 mM HEPES pH 7.9, 5 mM MgO^ 05 mM DTT, 6 M guanidinium hydrochloride) each. Afterwards, buffer C is four times diluted with an equal volume of buffer D (buffer C without guanidinium hydrochloride) leaving 50 ml of each dilution step for 5 min on the filter. The filter is washed twice in 50 ml buffer D and blocked in 50 ml buffer D supplemented with 5% nonfat dry milk for 30 min, After a short wash with 50 ml buffer D containing 0.25% nonfat dry milk the proteins are exposed to 6x10^ cpm/ml of a 5"-endlabeled synthetic oligonucleotide (DNASynthesizer 380 A, Applied Biosystcms) in 20 ml buffer D supplemented with 5 /lg/ml poly(dI-dC) and 0.25% nonfat dry milk for 120 min. After exposure to the DNA probe the filter is washed three times for 5 min in 100 ml buffer D containing 0.25% nonfat dry milt Thereafter, the filter U dried and exposed to Kodak X-Omat AR film for 48 hours at -70*C with the aid of an intensifying screen. The denaturarjon-renaturation cycle (3) used iu combination with short oligonucleotide probes containing one or more binding sites allows the detection of specific proteins in binding site blotting experiments with remarkably low background. This observation suggests that it is possible to map protein binding sites and also to determine the molecular weights of DNA binding proteins by using this method.