Abstract
Electrophoretic mobility shift assays (EMSA) have proven their usefulness for studying interactions between biological molecules. In the present protocol, a purified protein of interest is mixed with a 5′-end radiolabeled DNA probe. The bound complexes are separated by electrophoretic migration through a polyacrylamide gel and detected with a phosphorimager. The applications of EMSA are diverse, from thermodynamic and kinetic analyses to observation of bending and other conformational changes, stoichiometric inferences, or insights into cooperative protein binding.
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Abbreviations
- APS:
-
Ammonium persulfate
- ATP:
-
Adenosine triphosphate
- Bis-Tris:
-
1,3-bis(tris(hydroxymethyl)methylamino)propane
- BSA:
-
Bovine serum albumin
- cAMP:
-
3′-5′-cyclic adenosine monophosphate
- CAP:
-
E. coli cAMP receptor protein
- DNA:
-
Deoxyribonucleic acid
- DTT:
-
Dithiothreitol
- EDTA:
-
Ethylenediamine tetraacetic acid
- EMSA:
-
Electrophoretic mobility shift assay
- HEPES:
-
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- MOPS:
-
3-(N-morpholino)propanesulfonic acid
- PMSF:
-
Phenylmethylsulfonyl fluoride
- SELEX:
-
Systematic evolution of ligands by exponential enrichment
- SDS-PAGE:
-
Polyacrylamide gel electrophoresis carried in presence of sodium dodecyl sulfate
- TAE:
-
Tris-acetate-EDTA
- TBE:
-
Tris-borate-EDTA
- TE:
-
Tris-EDTA
- TEMED:
-
N,N,N′,N′-tetramethylethylenediamine
- Tris:
-
Tris(hydroxymethyl)aminomethane
References
Garner MM, Revzin A (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res 9:3047–3060
Hellman LM, Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions. Nat Protoc. doi:10.1038/nprot.2007.249
Lane D, Prentki P, Chandler M (1992) Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol Rev 56:509–528
Oxenburgh MS, Snoswell AM (1965) Use of Streptomycin in the Separation of Nucleic Acids from Protein in a Bacterial Extract. Nature. doi:10.1038/2071416a0
Cann JR (1989) Phenomenological theory of gel electrophoresis of protein-nucleic acid complexes. J Biol Chem 264:17032–17040
Vossen KM, Fried MG (1997) Sequestration stabilizes lac repressor-DNA complexes during gel electrophoresis. Anal Biochem 245:85–92
Fried M, Crothers DM (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res 9:6505–6525
Hope IA, Struhl K (1987) GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EMBO J 6:2781–2784
Kimsey HH, Waldor MK (2003) The CTX Repressor RstR Binds DNA Cooperatively to Form Tetrameric Repressor-Operator Complexes. J Biol Chem 279:2640–2647
Fried MG, Daugherty MA (1998) Electrophoretic analysis of multiple protein-DNA interactions. Electrophoresis 19:1247–1253
Orchard K, May GE (1993) An EMSA-based method for determining the molecular weight of a protein–DNA complex. Nucleic Acids Res 21:3335
Carey MF, Peterson CL, Smale ST (2013) Electrophoretic mobility-shift assays. Cold Spring Harb Protoc. doi:10.1101/pdb.prot075861
Haran TE, Mohanty U (2009) The unique structure of A-tracts and intrinsic DNA bending. Q Rev Biophys. doi:10.1017/S0033583509004752
Koo HS, Wu HM, Crothers DM (1986) DNA bending at adenine. thymine tracts. Nature 320:501–506
Zinkel SS, Crothers DM (1987) DNA bend direction by phase sensitive detection. Nature 328:178–181
Kim J, Zwieb C, Wu C, Adhya S (1989) Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector. Gene 85:15–23
Wu HM, Crothers DM (1984) The locus of sequence-directed and protein-induced DNA bending. Nature 308:509–513
Griffith J, Hochschild A, Ptashne M (1986) DNA loops induced by cooperative binding of lambda repressor. Nature 322:750–752
Liu-Johnson HN, Gartenberg MR, Crothers DM (1986) The DNA binding domain and bending angle of E. coli CAP protein. Cell 47:995–1005
Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822
Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510
Lee Y-Y, Barker CS, Matsumura P et al (2011) Refining the Binding of the Escherichia coli Flagellar Master Regulator, FlhD4C2, on a Base-Specific Level. J Bacteriol 193:4057–4068
Stoltenburg R, Reinemann C, Strehlitz B (2007) SELEX–a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol Eng 24:381–403
Sambrook J, Russel DW (2001) Molecular cloning : A laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York
Fried MG, Crothers DM (1984) Equilibrium studies of the cyclic AMP receptor protein-DNA interaction. J Mol Biol 172:241–262
Acknowledgement
This work was supported by the Fonds Québécois de la recherche sur la nature et les technologies (D.P.L.) and a Discovery Grant and Discovery Acceleration Supplement from the Natural Sciences and Engineering Council of Canada (V.B.). V.B. holds a Canada Research Chair in molecular bacterial genetics.
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Poulin-Laprade, D., Burrus, V. (2015). Electrophoretic Mobility Shift Assay Using Radiolabeled DNA Probes. In: Leblanc, B., Rodrigue, S. (eds) DNA-Protein Interactions. Methods in Molecular Biology, vol 1334. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2877-4_1
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DOI: https://doi.org/10.1007/978-1-4939-2877-4_1
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