Purification of Ribosome-Nascent-Chain Complex for Ribosome Profiling and Selective Ribosome Profiling

Abstract

Selective Ribosome Profiling (SeRP) is an emerging methodology, developed to capture cotranslational interactions in vivo. To date, SeRP is the only method that can directly capture, in near-codon resolution, ribosomes in action. Thus, SeRP allows us to study the mechanisms of protein synthesis and the network of protein-protein interactions that are formed already during synthesis. Here we report, in detail, the protocol for purification of ribosome- and Nascent-Chain associated factors, followed by isolation of ribosome-protected mRNA footprints, cDNA library generation and subsequent data analysis.

Keywords: Affinity purification; Cotranslational interactions; Nascent-chain; Ribosome; Ribosome occupancy.