The RNA of interest is modified with 5-bromo-UTP (BrUTP) by in vitro transcription, followed by incubation with a cell lysate (cytoplasmic extract or nuclear extract) to form the assembled RNA-RBP complexes. The BrU-labeled RNA-RBP complexes are then immunoaffinity-purified by anti-BrdU monoclonal antibody (mAb), which cross-reacts with BrUTP. RBPs associated with the BrU-labeled RNA can then be identified by immunoblotting or mass spectroscopy. Three different wash buffers provided in the RiboTrap Kit allow for analysis of both weakly and tightly bound RBPs. The wash buffer with mild conditions (Wash Buffer I) is usually used for primary screening of total RBPs, while wash buffer with stringent conditions (Wash Buffer II or III) is suitable for purifying tightly bound RBPs. Antisense RNA (corresponding to complementary RNA of interest) or truncated RNA of interest is used to exclude nonspecific binding proteins. Elution buffer composed of the optimal concentration of BrdU allows specific recovery of BrU-RNA/protein complexes. RBPs isolated with native conformations can be used in several downstream applications.