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

Methods. 2005 Dec;37(4):376-86. doi: 10.1016/j.ymeth.2005.07.018.


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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / metabolism
  • Cloning, Molecular
  • Cross-Linking Reagents / metabolism
  • Gene Amplification
  • Immunoprecipitation
  • Models, Biological
  • Molecular Biology / methods*
  • Nucleic Acid Amplification Techniques
  • Nucleic Acid Conformation / radiation effects
  • RNA Precursors / genetics
  • RNA Precursors / metabolism*
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / metabolism*
  • Ultraviolet Rays


  • Cross-Linking Reagents
  • RNA Precursors
  • RNA, Messenger
  • RNA-Binding Proteins