Abstract

INTRODUCTION

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.

METHODS

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.

RESULTS

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.

DISCUSSION

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.