Purification of RNA-Binding Proteins

Abstract

The majority of regulatory RNA sequences exert their function through interaction with proteins. Therefore, the identification of RNA-binding proteins is the key step in understanding the role of many RNA motifs. Here, we describe a straightforward method to identify RNA-binding proteins. In this approach, RNAs are immobilized on beads and incubated with protein lysates. After removing unbound fraction of proteins, the bound proteins are eluted by successive increasing of salt concentration. The lower the salt concentration, the lower is the binding affinity of a protein to the RNA. According to this principle, each elution fraction can contain a number of proteins which bind with similar affinity to the RNA. After gel electrophoretic separation and staining, each single protein band can be identified, isolated, and analyzed by mass spectrometry.