验证液-液相分离作为体外和人体细胞中无膜细胞器组装驱动力的方案。

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS

STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-23 DOI:10.1016/j.xpro.2024.103410

Marius Hedtfeld, Andrea Musacchio

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引用次数: 0

摘要

支架蛋白的液-液相分离（LLPS）常常被认为是驱动无膜细胞区室生物生成的因素。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。我们描述了在体外有拥挤剂或仿细胞介质的情况下检测 LLPS 的步骤。我们通过将蛋白质电穿孔到有丝分裂的 HeLa 细胞中的实验，对重组蛋白质的体外研究进行了补充。此外，我们还讨论了评估蛋白质定位和输送水平的步骤。有关本方案使用和执行的完整细节，请参阅 Hedtfeld 等人的文章1。

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Protocol for validating liquid-liquid phase separation as a driver of membraneless organelle assembly in vitro and in human cells.

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.¹.

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