Electrophoretic mobility shift assays

The electrophoretic mobility shift assay (EMSA), one of the most sensitive methods for studying the DNA-binding properties of a protein, can be used to deduce the binding parameters and relative affinities of a protein for one or more DNA sites or for comparing the affinities of different proteins for the same sites¹. It is also useful for studying higher-order complexes containing several proteins, observed as a 'supershift assay'. EMSA also can be used to study protein- or sequence-dependent DNA bending². In an EMSA, or simple 'gel shift', a ³²P-labeled DNA fragment containing a specific DNA site is incubated with a candidate DNA-binding protein. The protein-DNA complexes are separated from free (unbound) DNA by electrophoresis through a nondenaturing polyacrylamide gel. The protein retards the mobility of the DNA fragments to which it binds; thus, the free DNA migrates faster through the gel than does the DNA-protein complex. An image of the gel reveals the positions of the free and bound ³²P-labeled DNA.