Dual cross-linking ribonucleoprotein immunoprecipitation assay

Abstract

Ribonucleoprotein immunoprecipitation (RIP) is an antibody-based method to detect RNA–protein interactions in situ. In the assay, UV cross-linking is commonly used to preserve RNA–protein interactions for subsequent target identification. UV light is a zero-length cross linker and thus identifies proteins directly bound to RNAs. Here, we describe a dual cross-linking RIP method that involves sequential protein–protein cross-linking step with a protein–protein cross-linker, followed by protein–RNA fixation by UV irradiation. In this way, proteins that indirectly bound to RNA can be analyzed.