National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
Methods. 2011 Mar;53(3):285-94. doi: 10.1016/j.ymeth.2010.11.009. Epub 2010 Dec 7.
The results of cell and animal model studies demonstrate that molecular chaperones play an important role in controlling the processes of protein misfolding and amyloid formation in vivo. In addition, chaperones are involved in the appearance, propagation and clearance of prion phenotypes in yeast. The effect of chaperones on amyloid formation has been studied in great detail in recent years in order to elucidate the underlying mechanisms. An important approach is the direct study of effects of chaperones on amyloid fibril formation in vitro. This review introduces the methods and techniques that are commonly used to control and monitor the time course of fibril formation, and to detect interactions between chaperones and fibril-forming proteins. The techniques we address include thioflavin T binding fluorescence and filter retardation assays, size-exclusion chromatography, dynamic light scattering, and biosensor assays. Our aim in this review is to provide guidance on how to embark on study of the effect of chaperones on amyloid fibril formation, and how to avoid common problems that may be encountered, using examples and experience from the authors' lab and from the wider literature.
细胞和动物模型研究的结果表明，分子伴侣在控制体内蛋白质错误折叠和淀粉样形成过程中起着重要作用。此外，伴侣蛋白还参与了酵母中朊病毒表型的出现、传播和清除。近年来，为了阐明潜在机制，人们对伴侣蛋白对淀粉样纤维形成的影响进行了深入研究。一种重要的方法是直接研究伴侣蛋白对体外淀粉样纤维形成的影响。本综述介绍了常用的控制和监测纤维形成过程的方法和技术，以及检测伴侣蛋白与纤维形成蛋白之间相互作用的方法。我们所涉及的技术包括硫黄素 T 结合荧光和滤膜阻滞测定、凝胶过滤层析、动态光散射和生物传感器测定。我们的目的是通过作者实验室和更广泛文献中的示例和经验，为如何着手研究伴侣蛋白对淀粉样纤维形成的影响提供指导，并避免可能遇到的常见问题。
