Detecting RNA–protein interaction using end-labeled biotinylated RNA oligonucleotides and immunoblotting

Abstract

RNA–protein interaction can be detected by RNA pull-down and immunoblotting methods. Here, we describe a method to detect RNA–protein interaction using RNA pull down and to identify the proteins that are pulled- down by the RNA using immunoblotting. In this protocol, RNAs with specific sequences are biotinylated and immobilized onto Streptavidin beads, which are then used to pull down interacting proteins from cellular extracts. The presence of a specific protein is subsequently verified by SDS- polyacrylamide gel electrophoresis and immunoblotting with antibodies. Interactions between the SMN RNA and the PSF protein and between the caspase-2 RNA and the SRSF3 protein (SRp20) in nuclear extract prepared from HeLa cells are illustrated as examples.