Practice question

Demonstrating direct physical contact between protein and DNA in vitro is most convincingly accomplished by electrophoretic mobility shift. Purified protein incubated with labeled DNA probe produces retarded complex on native polyacrylamide gel due to increased mass and altered charge, indicating immediate binding without requiring crosslinking intermediates or additional factors. Chromatin Immunoprecipitation detects in vivo occupancy but may reflect indirect recruitment via multiprotein complexes. RIP assays RNA-protein interactions, RT-PCR quantifies transcripts. Thus EMSA provides definitive evidence of direct protein-DNA contact suitable for biochemical characterization.