Electrophoretic Mobility Shift Assay (EMSA) for Assessing RNA-Protein Binding and Complex Formation Using Recombinant...

Evaluating RNA-protein interactions is key to understanding post-transcriptional gene regulation. Electrophoretic mobility shift assays (EMSAs) remain a widely used technique to study these interactions, revealing information about binding affinities and binding modalities, including cooperativity and complex formation. Here, we detail, in a step-by-step protocol, how to perform EMSAs. We describe how to generate, purify, and quantitate ³²P-radiolabeled RNA by in vitro transcription, as well as the expression and purification of recombinant RNA-binding proteins in E. coli using ELAV as an example. We then describe how to set up binding reactions using serial dilutions in a microtiter plate format of recombinant ELAV and in vitro-transcribed RNA and how to perform EMSAs using native low-crosslinked acrylamide gels, with detailed graphically supported instructions and troubleshooting guides. Key features • Efficient production and purification of radiolabeled RNA probes via in vitro transcription and denaturing PAGE. • Reproducible binding assays shown using recombinant ELAV protein as an example. • Quantitative EMSA setup using serial dilutions in a microtiter plate for accurate binding curves. • Native gel preparation and optimized running conditions for high-resolution separation of RNA-protein complexes.