CLIP: A method for identifying protein-RNA interaction sites in living cells
CLIP: A method for identifying protein-RNA interaction sites in living cells
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DOI：
10.1016/j.ymeth.2005.07.018
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发表时间：
2005-12-01
期刊：
影响因子：
4.8
通讯作者：
Darnell, RB
中科院分区：
文献类型：
--
作者：
Ule, J;Jensen, K;Darnell, RB
核酸结合蛋白构成了所有功能注释的人类基因的四分之一。对于大多数这些蛋白质，尚未对蛋白质核酸接触的全基因组进行分析，从而限制了对蛋白质功能的全面理解的尝试。紫外线交联是一种通常用于确定蛋白质与核酸之间直接相互作用的位置的方法。我们已经开发了交联和免疫沉淀测定法，该测定法利用了共价蛋白核酸交联，使用免疫沉淀严格纯化了特定的蛋白-RNA复合物，然后进行SDS-PAGE分离。通过这种方式，可以去除与共免疫沉淀蛋白或珠子结合的绝大多数非特异性污染RNA。在这里，我们提出了一种改进的协议，该方案在SDS-PAGE步骤之前执行RNA接头连接，并描述其在NOVA在NOVA中的特定纯化和扩增NOVA在神经元中的应用。 （c）2005 Elsevier Inc.保留所有权利。
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. (c) 2005 Elsevier Inc. All rights reserved.