Electrophoretic Mobility Shift Assay Using Radiolabeled DNA Probes

Dominic Poulin-Laprade; Vincent Burrus

doi:10.1007/978-1-4939-2877-4_1

Electrophoretic Mobility Shift Assay Using Radiolabeled DNA Probes

Methods Mol Biol. 2015:1334:1-15. doi: 10.1007/978-1-4939-2877-4_1.

Authors

Dominic Poulin-Laprade¹, Vincent Burrus²

Affiliations

¹ Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada, J1K 2R1.
² Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada, J1K 2R1. Vincent.Burrus@USherbrooke.ca.

PMID: 26404140
DOI: 10.1007/978-1-4939-2877-4_1

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.

Keywords: DNA probe labeling; EMSA; Electrophoretic mobility shift assay; Gel retardation; Gel shift; Native polyacrylamide gel; Phosphorus-32; Protein–DNA interactions.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Probes / chemistry*
DNA Probes / genetics
DNA-Binding Proteins / chemistry
DNA-Binding Proteins / isolation & purification*
Electrophoretic Mobility Shift Assay / methods*
Protein Binding*
Radioisotopes

Substances

DNA Probes
DNA-Binding Proteins
Radioisotopes