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Methods. 2005 Dec;37(4):376-86.

CLIP: a method for identifying protein-RNA interaction sites in living cells.

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1
Howard Hughes Medical Institute and the Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, USA.

Abstract

Nucleic-acid binding proteins constitute nearly one-fourth of all functionally annotated human genes. Genome-wide analysis of protein-nucleic acid contacts has not yet been performed for most of these proteins, restricting attempts to establish a comprehensive understanding of protein function. UV cross-linking is a method typically used to determine the position of direct interactions between proteins and nucleic acids. We have developed the cross-linking and immunoprecipitation assay, which exploits the covalent protein-nucleic acid cross-linking to stringently purify a specific protein-RNA complex using immunoprecipitation followed by SDS-PAGE separation. In this way, the vast majority of non-specific contaminating RNA, which can bind to co-immunoprecipitated proteins or beads, can be removed. Here, we present an improved protocol that performs RNA linker ligation before the SDS-PAGE step, and describe its application to the specific purification and amplification of RNA ligands of Nova in neurons.

PMID:
16314267
DOI:
10.1016/j.ymeth.2005.07.018
[Indexed for MEDLINE]
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