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Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

Abstract

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, protein–nucleic 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 briefly described. References to extensions of the method and a troubleshooting guide are provided.

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Figure 1: Titration of a 214-bp lac promoter DNA fragment with Escherichia coli CAP protein.
Figure 2: Titration of a 16-residue single-stranded DNA with human AGT protein.
Figure 3: Titration of a 203-bp lac promoter DNA with Escherichia coli lactose repressor.
Figure 4: Titration of 1:1, 2:1 and 3:1 CAP-lac promoter complexes with lac repressor.

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Acknowledgements

This work was supported by the National Institutes of Health (NIH) grant GM-070662. The experiment shown in Figure 2 was performed by Dr. Joseph J. Rasimas. The experiment shown in Figure 4 was performed by Dr. J. Michael Hudson. We thank Drs. J.A. Stead and K.J. McDowall for communication of their results prior to publication.

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Hellman, L., Fried, M. Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions. Nat Protoc 2, 1849–1861 (2007). https://doi.org/10.1038/nprot.2007.249

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  • DOI: https://doi.org/10.1038/nprot.2007.249

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