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首页> 外文期刊>Nucleic Acids Research >Direct isolation of specific RNA-interacting proteins using a novel affinity medium
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Direct isolation of specific RNA-interacting proteins using a novel affinity medium

机译：使用新型亲和介质直接分离特定RNA相互作用蛋白

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摘要

Isolation of proteins that specifically interact with a given RNA or RNA regulation element is essential for studies on the molecular mechanisms of gene expression. Here, a novel method for direct isolation of such interacting proteins is described. It uses an affinity medium that consists of an interacting RNA with an artificially added 'tail', which is annealed to one end of a DNA 'arm', the other end of which is fixed covalently on the surface of aminosilanized glass powder. Thus the RNA itself is fully suspending, facilitating its interactions with proteins in its natural conformation. The proteins bound on the interacting RNA are eluted and subjected to SDS-PAGE, and the Coomassie-stained protein bands are cut and subjected to mass spectrometry (MS) analysis. Using this method, three proteins specifically interacting with the C/EBP beta 3'-untranslated region (3'-UTR) RNA were isolated and identified. This method is simple and convenient, and the DNA-glass powder medium can be used repeatedly.
机译：分离与给定RNA或RNA调控元件特异性相互作用的蛋白质对于研究基因表达的分子机制至关重要。在此，描述了一种直接分离这种相互作用蛋白的新颖方法。它使用一种亲和介质，该介质由相互作用的RNA和人工添加的“尾巴”组成，该尾巴退火至DNA“臂”的一端，另一端共价固定在氨基硅烷化玻璃粉末的表面上。因此，RNA本身可以完全悬浮，从而促进其与天然构象的蛋白质相互作用。洗脱结合在相互作用的RNA上的蛋白质，并进行SDS-PAGE，切割考马斯染色的蛋白质条带，并进行质谱（MS）分析。使用此方法，分离并鉴定了与C / EBP beta 3'-非翻译区（3'-UTR）RNA特异性相互作用的三种蛋白质。该方法简便易行，可重复使用DNA玻璃粉末培养基。

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