Bitan Gal, Teplow David B
Center for Neurologic Diseases, Brigham and Women's Hospital and Department of Neurology, Harvard Medical School, Boston, MA, USA.
Methods Mol Biol. 2005;299:3-9. doi: 10.1385/1-59259-874-9:003.
More than 20 diseases have been identified which are caused by the deposition of amyloid. Natural and chemically synthesized amyloidogenic proteins are used widely to study the structure, assembly, and physiologic effects of both oligomeric and fibrillar forms of these proteins. In many cases, conflicting results arise in these studies, in part owing to difficulties in reproducibly preparing amyloidogenic proteins in a well-defined assembly state. To avoid these problems, several methods have been devised that provide reliable means of preparing amyloid-forming proteins for experimental use. Here, we discuss methods that have been used successfully to prepare one such protein, the amyloid beta-protein (Abeta), involved in Alzheimer's disease. Methods for reproducible preparation of Abeta in a well-defined assembly state include isolation of low molecular weight (LMW) Abeta by size exclusion chromatography, filtration through LMW cut-off filters, and solubilization/lyophilization in the presence of reagents which facilitate disassembly of Abeta. These reagents include strong bases and acids, and fluorinated alcohols. These methods, which were originally developed for Abeta, are generally applicable to amyloidogenic peptides and proteins. In this chapter, we describe the preparation of LMW Abeta using size exclusion chromatography and filtration. The advantages and disadvantages of each method are discussed.
已经确定了20多种由淀粉样蛋白沉积引起的疾病。天然和化学合成的淀粉样蛋白生成蛋白被广泛用于研究这些蛋白的寡聚体和纤维状形式的结构、组装及生理效应。在许多情况下，这些研究中会出现相互矛盾的结果，部分原因是难以以明确界定的组装状态可重复地制备淀粉样蛋白生成蛋白。为避免这些问题，人们设计了几种方法，这些方法提供了可靠的手段来制备用于实验的淀粉样蛋白形成蛋白。在此，我们讨论已成功用于制备一种此类蛋白——参与阿尔茨海默病的β淀粉样蛋白（Aβ）——的方法。以明确界定的组装状态可重复制备Aβ的方法包括通过尺寸排阻色谱法分离低分子量（LMW）Aβ、通过低分子量截留滤器过滤，以及在有助于Aβ解聚的试剂存在下进行溶解/冻干。这些试剂包括强碱和强酸以及氟化醇。这些最初为Aβ开发的方法通常适用于淀粉样蛋白生成肽和蛋白。在本章中，我们描述使用尺寸排阻色谱法和过滤来制备LMW Aβ。并讨论了每种方法的优缺点。
