School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705, USA.
Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53705, USA.
STAR Protoc. 2021 Jan 22;2(1):100288. doi: 10.1016/j.xpro.2020.100288. eCollection 2021 Mar 19.
The discovery of potent cell-permeable E3 ubiquitin ligase ligands can significantly facilitate the development of proteolysis targeting chimeras (PROTACs). Here, we present a protocol to determine the binding affinity of ligands toward CRBN E3 ubiquitin ligase, using a cellular target engagement mechanism and in-cell ELISA assay. This protocol is easy to establish, with relatively low cost and rapid time frame. It can also be modified to measure the level of other proteins or determine the ligand affinity toward other E3s. For complete details on the use and execution of this protocol, please refer to Yang et al. (2020).
强效细胞渗透性 E3 泛素连接酶配体的发现，可以极大地促进蛋白水解靶向嵌合体（PROTACs）的发展。在这里，我们提出了一种使用细胞靶标结合机制和细胞内 ELISA 检测来测定配体与 CRBN E3 泛素连接酶结合亲和力的方案。该方案易于建立，成本相对较低，时间框架较短。它也可以进行修改，以测量其他蛋白质的水平或测定配体对其他 E3 的亲和力。有关该方案的使用和执行的完整详细信息，请参阅 Yang 等人（2020 年）。
