Preparation of aggregate-free, low molecular weight amyloid-beta for assembly and toxicity assays

Methods Mol Biol. 2005:299:3-9. doi: 10.1385/1-59259-874-9:003.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amyloid beta-Peptides / isolation & purification*
  • Biological Assay*
  • Humans
  • Time Factors

Substances

  • Amyloid beta-Peptides