Isolation of a sequence-specific RNA binding protein, polypyrimidine tract binding protein, using RNA affinity chromatography

Abstract

Many important cellular processes are mediated by sequence-specific RNA binding proteins, and it is often necessary to purify these proteins. When the RNA binding site is known, it is convenient to use this RNA as a matrix for affinity purification. The intronic splicing silencer (ISS) element present upstream of the N1 exon of the c-src pre-mRNA is a high-affinity binding site for the polypyrimidine tract binding protein (PTB). Using a 5′- biotinylated ISS RNA and PTB as an example, I describe a one-step method for affinity chromatography of RNA binding proteins from nuclear extracts. © 2008 Humana Press.