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DNA-Protein Interactions pp 1–15Cite as

Electrophoretic Mobility Shift Assay Using Radiolabeled DNA Probes

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1334))

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

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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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Authors and Affiliations

  1. Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada, J1K 2R1

    Dominic Poulin-Laprade & Vincent Burrus

Authors
  1. Dominic Poulin-Laprade
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  2. Vincent Burrus
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Corresponding author

Correspondence to Vincent Burrus .

Editor information

Editors and Affiliations

  1. Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada

    Benoît P. Leblanc

  2. Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada

    Sébastien Rodrigue

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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

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2876-7

  • Online ISBN: 978-1-4939-2877-4

  • eBook Packages: Springer Protocols

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