Regulation and Control of AP-1 Binding Activity in Embryotoxicity

The electrophoretic mobility shift assay (EMSA) is a sensitive relatively straightforward methodology used to detect sequence-specific DNA–protein interactions. It is the fundamental procedure of several variants that allow qualitative and quantitative assessments of protein–nucleic acid complexes. Classically, nuclear proteins and DNA are combined and the resulting mixture is electrophoretically separated in polyacrylamide or agarose gel under native conditions. The distribution within the gel is generally detected with autoradiography of the ³² P-labeled DNA. The underlying principle is that nucleic acid with protein bound to it will migrate more slowly through a gel matrix than the free nucleic acid. In this chapter, a representative protocol is described that addresses specific challenges of using whole embryos as the nuclear protein source, and the most common and informative EMSA variant, the “supershift,” is also presented. The important points are underscored and approaches for troubleshooting are explained. References are provided for alternative methods and extensions of the basic protocol.