Horizontal agarose gel mobility shift assay for protein-RNA complexes

Abstract

Recent advances in agarose gel electrophoresis protocols established conditions for the high-resolution separation of DNA and RNA using higher voltages combined with short run times. We subsequently developed a protocol for using these conditions to measure the binding affinity of a protein for an RNA ligand on an agarose gel. This native gel mobility shift assay is highly accessible, using common molecular biology reagents found in most laboratories. Here, we describe the protocol for carrying out native agarose gel electrophoresis to characterize the binding affinity of a protein for an RNA ligand. The electrophoresis time is less than 10 min, which minimizes the dissociation of protein and ligand. We have used the p19 siRNA binding protein and its cognate dsRNA ligand to demonstrate strategies for identifying optimal conditions to measure apparent binding constants using this agarose gel shift system.