Using fluorescence anisotropy to monitor chaperone dispersal of RNA-binding protein condensates.
Using fluorescence anisotropy to monitor chaperone dispersal of RNA-binding protein condensates.
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DOI：
10.1016/j.xpro.2022.101409
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发表时间：
2022-06-17
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热应激会触发出芽酵母中的一组特定蛋白质，形成固体状的生物分子凝聚体，并被分子伴侣分散。在这里，我们描述了一种使用生化重构和荧光各向异性来研究伴侣促进的凝聚物分散动力学的方案。虽然目前的方案是专门为研究聚(A)结合蛋白（Pab1）的热诱导冷凝物而设计的，但该方案可以进行修改，以研究任何在冷凝时表现出差异底物结合活性的蛋白质。有关使用和执行本协议的完整详情，请参阅。通过分子伴侣监测Pab1凝析物扩散的荧光各向异性数据分析量化凝析物最大扩散速率出版人注：开展任何实验方案都需要遵守当地机构的实验室安全和伦理指导方针。热应激会触发出芽酵母中的一组特定蛋白质，形成固体状的生物分子凝聚体，并被分子伴侣分散。在这里，我们描述了一种使用生化重构和荧光各向异性来研究伴侣促进的凝聚物分散动力学的方案。虽然目前的方案是专门为研究聚(A)结合蛋白（Pab1）的热诱导冷凝物而设计的，但该方案可以进行修改，以研究任何在冷凝时表现出差异底物结合活性的蛋白质。
Heat stress triggers a specific set of proteins in budding yeast to form solid-like biomolecular condensates, which are dispersed by molecular chaperones. Here, we describe a protocol to study the kinetics of chaperone-facilitated condensate dispersal using biochemical reconstitution and fluorescence anisotropy. Although the current protocol is tailored to study heat-induced condensates of poly(A)-binding protein (Pab1), the protocol can be modified to study any protein which shows differential substrate binding activity upon condensation. For complete details on the use and execution of this protocol, please refer to. Biochemical reconstitution of heat-induced Pab1 condensates Fluorescence anisotropy to monitor Pab1 condensate dispersal by molecular chaperones Data analysis to quantify the maximal rate of condensate dispersal Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Heat stress triggers a specific set of proteins in budding yeast to form solid-like biomolecular condensates, which are dispersed by molecular chaperones. Here, we describe a protocol to study the kinetics of chaperone-facilitated condensate dispersal using biochemical reconstitution and fluorescence anisotropy. Although the current protocol is tailored to study heat-induced condensates of poly(A)-binding protein (Pab1), the protocol can be modified to study any protein which shows differential substrate binding activity upon condensation.