Hedtfeld Marius, Musacchio Andrea
Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany; Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Essen, Germany.
STAR Protoc. 2024 Dec 20;5(4):103410. doi: 10.1016/j.xpro.2024.103410. Epub 2024 Oct 23.
Liquid-liquid phase separation (LLPS) of scaffold proteins has often been proposed to drive the biogenesis of membraneless cellular compartments. Here, we present a protocol to link in vitro LLPS propensity to localization in vivo. We describe steps for examining LLPS in vitro in the presence of crowding agents or cytomimetic media. We complement our in vitro studies with recombinant proteins with experiments of protein electroporation into mitotic HeLa cells. In addition, we discuss steps to assess protein localization and delivery levels. For complete details on the use and execution of this protocol, please refer to Hedtfeld et al..
支架蛋白的液-液相分离（LLPS）常被认为可驱动无膜细胞区室的生物发生。在此，我们提出了一种将体外LLPS倾向与体内定位联系起来的方案。我们描述了在拥挤剂或细胞模拟介质存在下体外检测LLPS的步骤。我们通过将重组蛋白电穿孔导入有丝分裂的HeLa细胞的实验，对我们的体外研究进行补充。此外，我们还讨论了评估蛋白质定位和递送水平的步骤。有关本方案使用和执行的完整详细信息，请参考赫德费尔德等人的研究。
