Electrophoretic Mobility Shift Assays for RNA–Protein Complexes

Abstract

The electrophoretic mobility shift assay (EMSA), or gel mobility shift assay, is a popular and powerful technique for the detection of RNA-protein interactions. It relies on the fact that naked RNA has certain mobility on nondenaturing gels, but if the RNA is bound by protein, the mobility of the RNA is reduced. Therefore, the binding of protein results in a characteristic upward shift of the RNA on a gel, as monitored using radiolabeled RNA. For reasons that are not completely understood, most RNA-protein complexes--particularly those that result from high-affinity interactions--do not dissociate during the prolonged times required for electrophoretic separation. Because high-affinity interactions are more stable, it is often possible to identify specific interactions over a "background" of weak interactions. Accordingly, EMSAs can be performed using complex mixtures of proteins such as cell extracts. They can be used to investigate a wide range of RNA-protein interactions--from single protein-binding events to assembly of large complexes such as the spliceosome. EMSAs can also be useful for determining kinetic parameters (such as affinity constants) for RNA-protein interactions.