Determination of the molecular weight of DNA-binding proteins using UV-crosslinking and SDS-PAGE

We describe the use of UV-crosslinking in combination with SDS-PAGE to determine the approximate molecular weight of DNA-binding proteins. A 5-bromo-2′-deoxyuridine (5-BrdU)-substituted, radioactively labeled double-stranded oligonucleotide representing the protein binding site is incubated with a crude nuclear extract containing the protein of interest. Following irradiation with a UV light source, the DNA/protein complex is subjected to SDS-PAGE and its molecular weight determined by comparison with appropriate protein standards.