An improved method for Southwestern blotting.

We have developed a modified Southwestern blotting technique which utilizes broad-spectrum protease inhibitors during nuclear protein extraction and a procedure for radiolabelling an oligonucleotide probe to a high specific activity. These modifications have resulted in minimal protein degradation during nuclear protein isolation and have permitted room temperature hybridizations, improving both the facility and sensitivity of the standard Southwestern assay. This technique was used in our laboratory to visualize NFAT-1 consensus sequence binding proteins in nuclear extracts of human promyelocytic HL-60 cells.