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Ammonium hydroxide treatment of Aβ produces an aggregate free solution suitable for biophysical and cell culture characterization

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Author(s): Timothy M. Ryan | Joanne Caine | Haydyn D.T. Mertens | Nigel Kirby | Julie Nigro | Kerry Breheney | Lynne J. Waddington | Victor A. Streltsov | Cyril Curtain | Colin L. Masters | Blaine R. Roberts

Journal: PeerJ
ISSN 2167-8359

Volume: 1;
Start page: e73;
Date: 2013;
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Keywords: Amyloid | Hexafluoroisopropanol | Ammonium Hydroxide | Aggregation | A β | A β toxicity | Oligomers

ABSTRACT
Alzheimer’s disease is the leading cause of dementia in the elderly. Pathologically it is characterized by the presence of amyloid plaques and neuronal loss within the brain tissue of affected individuals. It is now widely hypothesised that fibrillar structures represent an inert structure. Biophysical and toxicity assays attempting to characterize the formation of both the fibrillar and the intermediate oligomeric structures of Aβ typically involves preparing samples which are largely monomeric; the most common method by which this is achieved is to use the fluorinated organic solvent 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Recent evidence has suggested that this method is not 100% effective in producing an aggregate free solution. We show, using dynamic light scattering, size exclusion chromatography and small angle X-ray scattering that this is indeed the case, with HFIP pretreated Aβ peptide solutions displaying an increased proportion of oligomeric and aggregated material and an increased propensity to aggregate. Furthermore we show that an alternative technique, involving treatment with strong alkali results in a much more homogenous solution that is largely monomeric. These techniques for solubilising and controlling the oligomeric state of Aβ are valuable starting points for future biophysical and toxicity assays.
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