Assaying Cooperativity of ProteinDNA Interactions Using Agarose Gel Electrophoresis

DNA-binding proteins play essential roles in many cellular processes. Understanding on a molecular level how these proteins interact with their cognate sequences can provide important functional insights. Here, we describe a band shift assay in agarose gel to assess the mode of protein binding to a DNA molecule containing multiple protein-binding sites. The basis for the assay is that protein–DNA complexes display retarded gel electrophoresis mobility, due to their increased molecular weight relative to free DNA. The degree of retardation is higher with increasing numbers of bound protein molecules, thereby allowing resolution of complexes with differing protein–DNA stoichiometries. The DNA is radiolabeled to allow for visualization of both unbound DNA and all the different DNA–protein complexes. We present a quantitative analysis to determine whether protein binding to multiple sites within the same DNA molecule is independent or cooperative.