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

2. INTRODUCTION

Southwestern blotting, first described by Bowen and colleagues (1), is a powerful technique for identifying and characterizing DNA-binding proteins by their ability to bind to specific oligonucleotide probes. Nuclear protein extracts are typically separated electrophoretically on an SDS-polyacrylamide gel and then transferred to nitrocellulose for screening with oligonucleotide probes. The usefulness of this technique is hampered by its requirement for relatively large amounts of nuclear proteins (typically 50-100 mg), problems with protein degradation during isolation, and the difficulties in achieving efficient electrophoretic separation and transfer of a wide molecular size range of proteins.

Here, we describe a modified Southwestern technique that takes advantage of a highly efficiently labeled oligonucleotide probe for screening. In addition, the technique uses a wide-spectrum protease inhibitor that minimizes protein degradation and permits room temperature hybridization, and optimizes electrophoretic parameters for separation of a wide molecular size range of proteins. This technique was used in this laboratory to visualize NFAT-1 consensus sequence binding proteins in nuclear extracts of human promyelocytic HL-60 cells.