Analysis of DNA-protein interactions using proteins synthesized in vitro from cloned genes

To detect DNA binding activity, radiolabeled protein is incubated with specific DNA fragments, and protein-DNA complexes are separated from free protein by electrophoresis in native acrylamide gels. Unlike the more conventional mobility shift assay which utilizes (32)P-labeled DNA and unlabeled protein, the assay described here generally utilizes (35)S-labeled protein and unlabeled DNA. Major advantages of this method are that any desired mutant protein can be tested for its DNA-binding properties simply by altering the DNA template, and the subunit structure (e.g., dimer, tetramer) can be determined.