Drug affinity responsive target stability (DARTS) for small-molecule target identification.
Drug affinity responsive target stability (DARTS) for small-molecule target identification.
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DOI：
10.1007/978-1-4939-2269-7_22
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Huang, Jing
中科院分区：
文献类型：
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作者：
Pai, Melody Y;Lomenick, Brett;Hwang, Heejun;Schiestl, Robert;McBride, William;Loo, Joseph A;Huang, Jing
药物亲和性反应靶标稳定性(DARTS)是一种相对快速和直接的方法来识别潜在的小分子蛋白质靶标。它依赖于通过与小分子相互作用来保护目标蛋白质免受蛋白质降解的保护。这种方法的最大优点是能够使用天然的小分子，而不必固定或修饰它(例如，通过加入生物素、荧光、放射性同位素或光亲和性标记)。在这里，我们详细描述了使用复杂的蛋白质裂解物进行无偏见的DART以识别潜在的小分子结合靶标以及使用DART-Western blotting来测试、筛选或验证潜在的小分子靶标的方案。虽然这些想法主要是从我们和我们的合作者目前感兴趣的生物学领域的分子研究中发展出来的，但一般原则应该适用于对自然界中所有分子的分析。
Drug Affinity Responsive Target Stability (DARTS) is a relatively quick and straightforward approach to identify potential protein targets for small molecules. It relies on the protection against proteolysis conferred on the target protein by interaction with a small molecule. The greatest advantage of this method is being able to use the native small molecule without having to immobilize or modify it (e.g. by incorporation of biotin, fluorescent, radioisotope, or photo-affinity labels). Here we describe in detail the protocol for performing unbiased DARTS with complex protein lysate to identify potential binding targets of small molecules and for using DARTS-Western blotting to test, screen, or validate potential small molecule targets. Although the ideas have mainly been developed from studying molecules in areas of biology that are currently of interest to us and our collaborators, the general principles should be applicable to the analysis of all molecules in nature.