核酸
免疫沉淀
核糖核酸
生物化学
生物
RNA结合蛋白
蛋白质-蛋白质相互作用
DNA
邻近连接试验
连接器
分子生物学
计算生物学
化学
基因
计算机科学
操作系统
受体
作者
Jernej Ule,Kirk B. Jensen,Aldo Mele,Robert B. Darnell
出处
期刊:Methods
[Elsevier]
日期:2005-12-01
卷期号:37 (4): 376-386
被引量:541
标识
DOI:10.1016/j.ymeth.2005.07.018
摘要
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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