Immunoprecipitation and SDS-PAGE for Cross-Linking Immunoprecipitation (CLIP).

Abstract

This first part of this protocol is designed to optimize purification of the RNABP by immunoprecipitation for cross-linking immunoprecipitation (CLIP) experiments. The key variables to assess are the quality and quantity of antibody needed to immunoprecipitate most but not quite all of the RNABP (the titration will decrease nonspecific binding), and the tolerance of the antibody:antigen interaction to stringent wash conditions. The results of these experiments can be checked first by western blot, and subsequently using the pilot CLIP protocol described in the second half of this protocol. RNase-treated cross-linked RNABP:RNA complexes from mixed lysates or cell pellets are immunoprecipitated using conditions optimized in the first half of the protocol. 3' Linkers are added, the RNA is radiolabeled, and the complexes are purified on SDS-PAGE. The pilot experiment will identify the optimal RNase concentration for the particular sample and will assess the quality and purity of the RNABP-RNA complexes following labeling of the RNA tags with 32P. This can be done without ligation of the 3' linker as described in the main protocol below. The pilot experiment assesses whether sufficient RNA-protein complexes can be detected by autoradiography and whether contaminating RNA ligands are present in immunoprecipitations compared with control samples. Once it is confirmed that the signal-to-noise ratio for detection of RNA-protein complexes after immunoprecipitation is sufficient, the optimal immunoprecipitation conditions should be incorporated into the general CLIP protocol including the steps of cross-linking, RNase digestion, linker ligation, and labeling of RNA "tags," and the results analyzed by autoradiography.