Jeffrey S. Handen and Helene F. Rosenberg

The Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

Received 6/4/97; Accepted 6/10/97

4. RESULTS AND DISCUSSION

A number of modifications to the Southwestern blotting was done to increase the sensitivity of detection without excessive background and with the improved ease of room temperature hybridization. End-labeling with a 15 fold excess of [g-³²P]ATP followed by size-selection created a radiolabeled probe with high specific activity that yielded little background into the room temperature-hybridization step. Room temperature hybridization steps were made possible by the inclusion of the protease inhibitor cocktail in the nuclear sample preparation. We found this method allowed detection of an approximately 90 kDa DNA binding protein on a nitrocellulose blot of HL-60 nuclear proteins that could not be detected using the less efficiently labeled probe (Figures 1A and 1B). Additionally, a faint band of approximately 100 kDa was also detected. Using this method, we were able to detect strong signals from as little as 5 mg of protein from a crude nuclear extract after an overnight exposure to X-ray film.

Figure 1: (A) Southwestern blot of HL-60 nuclear extract probed with a double-stranded oligonucleotide containing an NFAT-1 consensus binding site that was end-labeled by standard techniques (7). (B) Southwestern blot of HL-60 nuclear extract probed with the same double-stranded oligonucleotide end-labeled to a 10-fold greater specific activity. Lane 1, 5 mg nuclear protein extract; Lane 2, 12.5 mg nuclear protein extract; Lane 3, 25 mg nuclear protein extract. Arrow points to an additional minor band (100 kDa).