Department of Botany and Pathology, Purdue University, West Lafayette, IN, USA.
Center for Plant Biology, Purdue University, West Lafayette, IN, USA.
Methods Mol Biol. 2021;2213:175-182. doi: 10.1007/978-1-0716-0954-5_15.
Drug affinity responsive target stability (DARTS) assay is used to detect the interaction between a ligand and a protein based on the observation that some ligands can protect the target protein from degradation by proteases when mixed in a solution. To set up the assay, a ligand is first mixed with a purified candidate target protein or a total cell lysate that contains a candidate target protein. Then, different amounts of protease are added to the mixture to allow the enzyme to digest the protein in the mixture. After protease digestion, the candidate target protein is detected by assays such as western blot, silver staining, or Coomassie blue staining. In theory, the candidate protein should be protected by the ligand from protease digestion, which is reflected by higher abundance of the candidate protein in mixtures containing the ligand compared with the control treatment. There are a few significant advantages of DARTS: (a) the ligand does not need to be modified so the native ligand could be used; (b) the candidate target protein could be either purified protein or protein that is present in the total cell lysate; and (c) the assay can be used together with proteomics analysis to identify an unknown target protein. The assay is especially valuable to test the interaction between the ligand and membrane proteins that are often challenging to purify. In this chapter, we use Endosidin2 (ES2) and its target protein Arabidopsis thaliana EXO70A1 (AtEXO70A1) as an example to show the step-by-step procedure of the DARTS assay.
药物亲和反应靶标稳定性（DARTS）测定法用于检测配体与蛋白质之间的相互作用，其依据是观察到一些配体在混合于溶液中时可以保护靶蛋白免受蛋白酶的降解。为了建立该测定法，首先将配体与纯化的候选靶蛋白或包含候选靶蛋白的总细胞裂解物混合。然后，向混合物中加入不同量的蛋白酶，以允许酶消化混合物中的蛋白质。蛋白酶消化后，通过 Western blot、银染或考马斯亮蓝染色等测定法检测候选靶蛋白。从理论上讲，候选蛋白应该受到配体的保护而免受蛋白酶的消化，这反映在含有配体的混合物中的候选蛋白比对照处理中的丰度更高。DARTS 具有几个显著的优点：(a) 配体不需要修饰，因此可以使用天然配体；(b) 候选靶蛋白可以是纯化的蛋白，也可以是总细胞裂解物中存在的蛋白；(c) 该测定法可与蛋白质组学分析一起使用，以鉴定未知的靶蛋白。该测定法特别有价值，可以测试配体与膜蛋白之间的相互作用，而膜蛋白通常难以纯化。在本章中，我们以 Endosidin2 (ES2)及其靶蛋白 Arabidopsis thaliana EXO70A1 (AtEXO70A1)为例，展示 DARTS 测定法的逐步步骤。
