Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
STAR Protoc. 2021 Feb 1;2(1):100308. doi: 10.1016/j.xpro.2021.100308. eCollection 2021 Mar 19.
To date, phase separation studies have largely been limited to assays using non-native conditions and aggregation-prone recombinant proteins that are often difficult to purify. This protocol describes the determination of relative protein concentration thresholds for phase separation through fluorescent imaging of GFP-tagged proteins in cells. The commercial availability of various plasmids and antibodies, as well as advances in gene editing, allow this procedure to be modified for the study of various phase-separating proteins in their relevant contexts. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020).
迄今为止，相分离研究主要局限于使用非天然条件和聚集倾向重组蛋白的检测，这些蛋白通常难以纯化。本方案描述了通过 GFP 标记的蛋白在细胞中的荧光成像来确定相分离的相对蛋白浓度阈值。各种质粒和抗体的商业可用性以及基因编辑技术的进步，使得该程序可以针对各种相关相分离蛋白进行修改。如需了解本方案的详细使用和执行情况，请参考 Lee 等人（2020 年）。
