The detection of DNA-binding proteins by protein blotting

Abstract

A method, called “protein blotting,” for the detection of DNA-binding proteins is described. Proteins are separated on an SDS-polyacrylamide gel. The gel ia sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replicais used to detect DNA-binding proteins, RKA-binding proteins or histonebinding proteins by incubation of the filter with [³²P]DNA, [¹²⁵I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed.