Payliss Brandon J, Wyatt Haley D M
Department of Biochemistry, University of Toronto, Toronto, ON M56 1A8, Canada.
Department of Biochemistry, University of Toronto, Toronto, ON M56 1A8, Canada; Canada Research Chairs Program, Temerty Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada.
STAR Protoc. 2023 Mar 13;4(2):102152. doi: 10.1016/j.xpro.2023.102152.
Phosphorylation is a post-translational modification that can alter protein structure and regulate protein-protein interactions. Here, we present a procedure for in vitro phosphorylation of the MUS81-binding region of SLX4 (SLX4) using cyclin-dependent kinase 1-cyclin B. We describe steps for the dialysis and phosphorylation of target proteins followed by purification using size-exclusion chromatography. Finally, we detail a system to monitor phosphorylation effectiveness and identify phosphorylated residues. We anticipate this protocol to be readily adapted for other protein targets or kinases. For complete details on the use and execution of this protocol, please refer to Payliss et al. (2022)..
磷酸化是一种翻译后修饰，可改变蛋白质结构并调节蛋白质-蛋白质相互作用。在此，我们展示了一种使用细胞周期蛋白依赖性激酶1-细胞周期蛋白B对SLX4（SLX4）的MUS81结合区域进行体外磷酸化的方法。我们描述了靶蛋白的透析和磷酸化步骤，随后使用尺寸排阻色谱法进行纯化。最后，我们详细介绍了一个监测磷酸化效果并鉴定磷酸化残基的系统。我们预计该方案可轻松适用于其他蛋白质靶标或激酶。有关该方案的使用和执行的完整详细信息，请参考Payliss等人（2022年）的文献。
