Silica-based solid-phase extraction of cross-linked nucleic acid-bound proteins.
Silica-based solid-phase extraction of cross-linked nucleic acid-bound proteins.
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DOI：
10.26508/lsa.201800088
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发表时间：
2018-06
影响因子：
4.4
通讯作者：
Hentze MW
中科院分区：
文献类型：
--
作者：
Asencio C;Chatterjee A;Hentze MW
2C方法允许快速和直接分离核酸-蛋白质复合物，大大简化了DNA和RNA-蛋白质相互作用研究的下游应用。蛋白质与核酸相互作用以调节细胞功能。这些调控相互作用的研究往往受到当前方案分离相关核酸-蛋白质复合物的有限效率的阻碍。在这份报告中，我们描述了一个快速和简单的程序，高度富集交联的核酸结合的蛋白质，被称为“2C”的“复杂的捕获”。该方法基于以下观察结果：用于核酸纯化的基于二氧化硅基质的柱也有效地保留UV交联的核酸-蛋白质复合物。作为原理的证明，2C用于从酵母和哺乳动物Huh 7细胞分离RNA结合蛋白。2C方法使RNA标记变得多余，并且可以通过Western印迹法观察和验证特定的RNA-蛋白质相互作用。通过2C分离的RNA-蛋白质复合物随后可以被免疫沉淀，这表明2C原则上与敏感的下游应用相容。我们认为2C可以大大简化核酸-蛋白质相互作用的研究，并使DNA和RNA生物学领域的研究人员受益。
The 2C method allows the rapid and straightforward isolation of nucleic acid–protein complexes, greatly simplifying downstream applications for the study of DNA– and RNA–protein interactions. Proteins interact with nucleic acids to regulate cellular functions. The study of these regulatory interactions is often hampered by the limited efficiency of current protocols to isolate the relevant nucleic acid–protein complexes. In this report, we describe a rapid and simple procedure to highly enrich cross-linked nucleic acid–bound proteins, referred to as “2C” for “complex capture.” This method is based on the observation that silica matrix–based columns used for nucleic acid purification also effectively retain UV cross-linked nucleic acid–protein complexes. As a proof of principle, 2C was used to isolate RNA-bound proteins from yeast and mammalian Huh7 cells. The 2C method makes RNA labelling redundant, and specific RNA–protein interactions can be observed and validated by Western blotting. RNA–protein complexes isolated by 2C can subsequently be immunoprecipitated, showing that 2C is in principle compatible with sensitive downstream applications. We suggest that 2C can dramatically simplify the study of nucleic acid–protein interactions and benefit researchers in the fields of DNA and RNA biology.
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影响因子：
16.6
作者：
Beckmann BM;Horos R;Fischer B;Castello A;Eichelbaum K;Alleaume AM;Schwarzl T;Curk T;Foehr S;Huber W;Krijgsveld J;Hentze MW
通讯作者：
Hentze MW
影响因子：
14.8
作者：
Castello, Alfredo;Horos, Rastislav;Hentze, Matthias W.
通讯作者：
Hentze, Matthias W.
影响因子：
2.9
作者：
CHOMCZYNSKI, P;SACCHI, N
通讯作者：
SACCHI, N
影响因子：
13.8
作者：
Orlando, V
通讯作者：
Orlando, V
影响因子：
7.7
作者：
Battaglia, Sofia;Lidschriber, Michael;Cramer, Patrick
通讯作者：
Cramer, Patrick