In this video, we describe the systematic evolution of ligands by the exponential enrichment (SELEX) method to identify specific RNA-binding sequences for a target protein of interest. The protein is incubated with a large pool of randomized RNA sequences, and the protein-binding RNA sequences are isolated, PCR-amplified, and sequenced to identify their protein-binding sites.
Protocol
1. Protein binding reaction and separation of bound RNA Carry out binding of protein and RNA in 10 mM Tris-HCl, pH 7.5 in a volume of 100 µL by adding the following ingredients to these final concentrations: 50 mM KCl, 1 mM DTT, 0.09 µg/µL bovine serum albumin, 0.5 units/µL RNasin, 0.15 µg/µL tRNA, 1 mM EDTA, and 30 µL of appropriate recombinant protein (PTB) concentration. Add RNA from the appropriate pool. NOTE: The splicing factor …