A User's Guide for Phase Separation Assays with Purified Proteins.
A User's Guide for Phase Separation Assays with Purified Proteins.
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DOI：
10.1016/j.jmb.2018.06.038
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发表时间：
2018-11-02
影响因子：
5.6
通讯作者：
Hyman AA
中科院分区：
文献类型：
--
作者：
Alberti S;Saha S;Woodruff JB;Franzmann TM;Wang J;Hyman AA
通过蛋白质相分离形成无膜细胞器和区室是细胞组织细胞质和核质的重要方式。使用纯化蛋白质进行体外相分离测定已成为研究形成无膜区室的蛋白质的标准方法。到目前为止，各种蛋白质已经被纯化并测试了它们在体外相分离和形成液体冷凝物的能力。然而，相分离蛋白通常容易聚集并且难以纯化和处理。因此，相分离测定的结果通常因实验室而异，并且不易重现。因此，迫切需要高质量的蛋白质、标准化程序以及普遍认可的蛋白质纯化和相分离测定实践。本文提供了蛋白质纯化方案，并指导用户了解体外蛋白质相分离测定的实用性，包括最佳实践方法和要避免的陷阱。我们相信，这份协议和实践概要将为研究蛋白质相行为的科学家提供有用的资源。无膜细胞器通过相分离形成。无膜细胞器可以用最少的成分重建。相分离蛋白质难以纯化和处理。我们提供使用相分离蛋白质的指南和方案。
The formation of membrane-less organelles and compartments by protein phase separation is an important way in which cells organize their cytoplasm and nucleoplasm. In vitro phase separation assays with purified proteins have become the standard way to investigate proteins that form membrane-less compartments. By now, various proteins have been purified and tested for their ability to phase separate and form liquid condensates in vitro. However, phase-separating proteins are often aggregation-prone and difficult to purify and handle. As a consequence, the results from phase separation assays often differ between labs and are not easily reproduced. Thus, there is an urgent need for high-quality proteins, standardized procedures, and generally agreed-upon practices for protein purification and conducting phase separation assays. This paper provides protocols for protein purification and guides the user through the practicalities of in vitro protein phase separation assays, including best-practice approaches and pitfalls to avoid. We believe that this compendium of protocols and practices will provide a useful resource for scientists studying the phase behavior of proteins. Membrane-less organelles form by phase separation. Membrane-less organelles can be reconstituted from minimal components. Phase-separating proteins are difficult to purify and handle. We provide guidelines and protocols for working with phase-separating proteins.
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