[25] Protein RNA-binding activity measured by northwestern blot analysis: The interferon-inducible RNA-dependent protein kinase PKR

The chapter describes the procedure for the analysis of RNA-binding activity by the Northwestern RNA blot assay. The procedure is described with the human RNA-dependent protein kinase (PKR). The Northwestern RNA blot assay provides an efficient approach for the identification of regions of a protein responsible for its RNA-binding activity. The strategy for the measurement of RNA-binding activity by Northwestern analysis involves the immobilization of target proteins on a filter membrane. Proteins fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) are electroblotted onto a nitrocellulose filter membrane by standard techniques. The fiber-bound proteins are then analyzed for RNA-binding activity using a radioactive RNA probe; this RNA-protein blot analysis constitutes the Northwestern assay. Subsequently, a Western immunoblot analysis is carried out using the same filter membrane as is used for the Northwestern analysis to verify that comparable amounts of test proteins are present. The Western analysis is especially important in the cases of proteins that do not register as RNA-binding proteins in the Northwestern assay.