Northwestern Blot Analysis: Detecting RNA–Protein Interaction After Gel Separation of Protein Mixture

Northwestern assays detect a direct binding of a given RNA molecule to a protein immobilized on a nitrocellulose membrane. Here, we describe protocols to prepare 32P-labeled RNA probes and to use them to assay for RNA–protein interactions after partially purified protein preparations are resolved on denaturing SDS-polyacrylamide gels. The method can unambiguously determine whether the protein of interest can directly and independently bind RNA even in the presence of contaminating bacterial proteins or degradation products that at times may hinder interpretation of results obtained from gel mobility shift or RNP immunoprecipitation assays.