Wang Hui, Zhou Rong, Ji Xiong
Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China; Department of Pathogenic Biology, Chengdu Medical College, Chengdu 610500, China.
Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
STAR Protoc. 2023 Mar 28;4(2):102202. doi: 10.1016/j.xpro.2023.102202.
Clustering of RNA polymerase II carboxy-terminal domain (CTD) and CTCF DNA-binding domains (DBDs) have been viewed as emerging mechanisms of transcription regulation and three-dimensional chromatin organization. In this protocol, we address the need for a quantitative means of investigating phase-separation mechanisms of Pol II transcription and CTCF functioning. We describe steps for protein purification, droplet formation, and automeasuring droplet properties. We then detail quantification during Pol II CTD and CTCF DBD clustering and outline their limitations. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022) and Zhou et al. (2022)..
RNA聚合酶II羧基末端结构域（CTD）和CTCF DNA结合结构域（DBD）的聚集已被视为转录调控和三维染色质组织的新兴机制。在本方案中，我们满足了对一种定量方法的需求，该方法用于研究Pol II转录和CTCF功能的相分离机制。我们描述了蛋白质纯化、液滴形成和自动测量液滴特性的步骤。然后，我们详细介绍了Pol II CTD和CTCF DBD聚集过程中的定量方法，并概述了它们的局限性。有关本方案使用和执行的完整详细信息，请参阅Wang等人（2022年）和Zhou等人（2022年）的文献。
