Protein Complex Binding to Promoter DNA: Immobilized Template Assay

Abstract

INTRODUCTIONLarge multiprotein complexes involved in transcription generally cannot be resolved by the methods conventionally used to study cooperative DNA binding by smaller proteins. The immobilized template recruitment assay can be used to circumvent this limitation. In this procedure, a biotinylated DNA fragment is immobilized on streptavidin-coated magnetic beads. DNA-binding proteins are incubated with the immobilized template, and proteins not specifically assembled in a complex are removed by separating the immobilized template with a magnetic particle concentrator and subsequent washing of the magnetic bead/template pellet. Bound proteins are detected by immunoblotting. To detect cooperative binding, subsaturating amounts of two or more proteins or protein complexes are added to the immobilized template. Template binding of a protein in concert with its cooperative binding partner is then compared to the binding of each individual protein or protein complex alone. In addition to studying the binding of protein complexes, the immobilized promoter template can also be used in functional assays such as in vitro transcription, allowing a correlation of complex assembly with function.