Gel Mobility Shift Assays to Detect ProteinRNA Interactions

The gel mobility shift assay is a powerful technique for detecting and quantifying protein–RNA interactions. While other techniques such as filter binding and isothermal titration calorimetry (ITC) are available for quantifying protein–RNA interactions, gel shift analysis provides the added advantage that you can visualize the protein–RNA complexes. In the gel shift assay, protein–RNA complexes are typically separated from the unbound RNA using native polyacrylamide gels in Tris/borate/EDTA buffer, although an alternative Tris-glycine buffering system is superior in many situations. Here, we describe both gel shift methods, along with strategies to improve separation of protein–RNA complexes from free RNA, which can be a particular challenge for small RNA binding proteins.