CLIP: A method for identifying protein-RNA interaction sites in living cells
Date
2005
Authors
Ule, J.
Jensen, K.
Mele, A.
Darnell, R.
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Type:
Journal article
Citation
Methods, 2005; 37(4):376-386
Statement of Responsibility
Jernej Ule, Kirk Jensen, Aldo Mele and Robert B. Darnell
Conference Name
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.
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http://www.elsevier.com/wps/find/journaldescription.cws_home/622914/description#description