Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions.
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.
Hellman
Lance M
LM
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0509, USA.
Fried
Michael G
MG
D001426:Bacterial Proteins / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D006801:Humans; D007763:Lac Operon / Q000235:genetics; D056968:Lac Repressors; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016601:RNA-Binding Proteins / Q000032:analysis*; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C481662:crp protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase
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