NDLI: CLIP (cross-linking and immunoprecipitation) identification of RNAs bound by a specific protein.

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CLIP (cross-linking and immunoprecipitation) identification of RNAs bound by a specific protein.

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CLIP (cross-linking and immunoprecipitation) identification of RNAs bound by a specific protein.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Darnell, Robert
Abstract	Guilt by association remains a powerful argument for assigning function in biological processes: Proteins that bind a specific RNA are strongly implicated in modulating the fate and action of that RNA. Detecting such associations is a major goal in understanding RNA function. The cross-linking and immunoprecipitation (CLIP) method described here allows assignments of RNA-binding proteins to their RNA targets by using ultraviolet (UV) cross-linking to covalently capture close (near-covalent bond distances) association of RNA with protein, followed by immunopurification of the protein partner with extraction and subsequent characterization of the RNA partner sequence(s). Pools of target sequences can be captured and their genomic origins are studied so that regulatory and other functional relationships between the protein and its RNA targets can be inferred. The advantage of this protocol is that it captures only intimately associated RNAs and proteins, and so is expected to be highly specific. Some investigators report capturing cross-links between rRNA and nonribosomal proteins in cases where ribosomes are not efficiently cleared from the lysate before immunopurification of the target protein, so the limits of specificity for the method are yet to be fully explored. Its main disadvantages are its many steps and that it may fail to work for all proteins or capture all legitimate targets of any given protein if the necessary chemical groups are not optimally arranged for photocross-linking in the complex. However, properly performed, this method should provide many useful insights into the nature of the RNA ligands for an RNA-binding protein of interest.
File Format	HTM / HTML
ISSN	19403402
Issue Number	11
Journal	Cold Spring Harbor Protocols
Volume Number	2012
e-ISSN	15596095
Language	English
Publisher	Cold Spring Harbor Laboratory Press
Publisher Date	2012-11-01
Publisher Place	United States
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Discipline Clinical Laboratory Techniques Immunoprecipitation Methods Rna-binding Proteins Metabolism Rna Genetics Isolation & Purification Cross-linking Reagents Journal Article
Content Type	Text
Resource Type	Article
Subject	Biochemistry, Genetics and Molecular Biology

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RNA immunoprecipitation to determine RNA-protein associations in vivo.

Ultraviolet photochemical cross-linking to detect RNA-binding proteins.

A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins.

Dual cross-linking ribonucleoprotein immunoprecipitation assay.

CLIP: construction of cDNA libraries for high-throughput sequencing from RNAs cross-linked to proteins in vivo.

Unbiased identification of protein-bait interactions using biochemical enrichment and quantitative proteomics.

Experimental strategies for the identification of DNA-binding proteins.

RNase footprinting to map sites of RNA-protein interactions.

Reverse footprinting to map sites of RNA-protein interactions.

CLIP (cross-linking and immunoprecipitation) identification of RNAs bound by a specific protein.

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