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Electrophoretic mobility shift assay (EMSA) for detecting proteinnucleic acid interactions
Lance M Hellman & Michael G Fried
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Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0509, USA. Correspondence should be addressed to M.G.F. ([email protected]).
Published online 26 July 2007; doi:10.1038/nprot.2007.249
The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, proteinnucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briey described. References to extensions of the method and a troubleshooting guide are provided.
INTRODUCTIONThe electrophoresis mobility shift assay (EMSA) is a rapid and sensitive method to detect proteinnucleic acid interactions16. It is based on the observation that the electrophoretic mobility of a proteinnucleic acid complex is typically less than that of the free nucleic acid (Fig. 1). The current, widely-used assay differs little from that originally described by Fried and Crothers7 and Garner and Revzin8; although precursors to the technique can be found in the earlier literature911. Mobility-shift assays are often used for qualitative purposes; although under appropriate conditions they can provide quantitative data for the determination of binding stoichiometries, afnities and kinetics3,6,12. Methods used in per
forming the assay differ for each purpose, and a large number of variants have been described in the literature (see Table 1).
0 Advantages and limitations of EMSAThe mobility shift assay has a number of strengths. The basic technique is simple to perform, yet it is robust enough to accommodate a wide...