Electrophoretic Mobility Shift Assay (EMSA) for Assessing RNA–Protein Binding and Complex Formation Using Recombinant RNA-Binding Proteins and In Vitro–Transcribed RNA

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.