Separation and Characterization of Protein-DNA Complexes by EMSA and In-Gel Footprinting

Abstract

In-gel footprinting enables the precise identification of protein binding sites on the DNA after separation of free and protein-bound DNA molecules by gel electrophoresis in native conditions and subsequent digestion by the nuclease activity of the 1,10-phenanthroline-copper ion [(OP)₂-Cu⁺] within the gel matrix. Hence, the technique combines the resolving power of protein-DNA complexes in the electrophoretic mobility shift assay (EMSA) with the precision of target site identification by chemical footprinting. This approach is particularly well suited to characterize distinct molecular assemblies in a mixture of protein-DNA complexes and to identify individual binding sites within composite operators, when the concentration-dependent occupation of binding sites, with a different affinity, results in the generation of complexes with a distinct stoichiometry and migration velocity in gel electrophoresis.

Keywords: 1,10-phenanthroline; Chemical nucleases; Denaturing gel electrophoresis; Electrophoretic mobility shift assay; In-gel footprinting; Native gel electrophoresis; Protein–DNA binding; Sequencing ladders.