Principles and problems of the electrophoretic mobility shift assay

Holden, Neil S. and Tacon, Claire E. (2011) Principles and problems of the electrophoretic mobility shift assay. Journal of Pharmacological and Toxicological Methods, 63 (1). pp. 7-14. ISSN 1056-8719

Full content URL:


Request a copy
[img] PDF
__network.uni_staff_S2_jpartridge_1-s2.0-S1056871910000456-main.pdf - Whole Document
Restricted to Repository staff only

Item Type:Article
Item Status:Live Archive



The electrophoretic mobility shift assay (EMSA) is classically used to detect DNA binding proteins, the tenet of the EMSA is that DNA with protein bound, migrates through a polyacrylamide gel more slowly than the corresponding free unbound DNA.


The classical EMSA protocol has 4 major steps: 1) The isolation of proteins from cells. Since the vast majority of active DNA binding proteins are present within the nucleus, a sequential membrane lysis protocol is used which yields purified nuclear protein. 2) Manufacture and radiolabelling of the DNA probe. Phosphorous 32 ((32)P) is attached to the 5' ends of the DNA probe through use of (32)P-γATP as a substrate for T4 polynucleotide kinase. DNA probes can both be purchased or custom made. 3) Purified proteins and radiolabelled DNA probes are co-incubated with an EMSA binding buffer to promote binding of the proteins with the DNA probe. If a supershift EMSA is being carried out, the reaction also contains a selective antibody which when bound to the protein-DNA complexes, causes further retardation within the gel. 4) The DNA-protein complexes are loaded and run on a non-denaturing polyacrylamide gel causing separation of the DNA-protein complexes from the free DNA probes. The polyacrylamide gels are then dried down and analysed via autoradiography.


As a demonstration of the effectiveness of this protocol, we show that tumour necrosis factor (TNF)α and phorbol 12-myristate 13-acetate (PMA) stimulation of A549 cells, results in a number of DNA-protein complexes being induced when compared to untreated cells. We also demonstrate that these complexes contain the p50 and p65 subunits of NF-κB through utilisation of the EMSA supershift protocol.


We provide detailed troubleshooting hints and tips for this technique and discuss the limitations of the EMSA, as well as a number of EMSA variants and alternative techniques.

Keywords:EMSA, Troubleshooting, End-labelling, Radiolabelled, DNA, Supershift
Subjects:C Biological Sciences > C700 Molecular Biology, Biophysics and Biochemistry
C Biological Sciences > C500 Microbiology
Divisions:College of Science > School of Life Sciences
ID Code:15137
Deposited On:21 Oct 2014 14:41

Repository Staff Only: item control page