Protocol for quantitative and qualitative analyses of the in vitro aggregation of synthetic beta-amyloid. A method applicable to the identification of substances that may have therapeutic efficacy for Alzheimer's disease.
Biochemical studies of beta-amyloid (Abeta) aggregation have been hampered by the lack of a simple method to examine simultaneously the extent of aggregation and the structural nature of the aggregates. Consequently, often only the extent of aggregation is analyzed and reported. This means that there is often no knowledge of whether the aggregates consist of fibrils, a structure crucial for their neurotoxicity. Here we describe the development of a protocol for quantitatively and qualitatively evaluating the in vitro aggregation of synthetic Abeta. Specifically, a fluorescein derivatized Abeta(1-42) peptide (FITC-Abeta(1-42)) was used as the aggregation material. We found that the fluorescent Abeta peptide aggregated as efficiently as the native ones and the extent of their aggregation could be determined accurately by fluorescence spectrophotometry. Significantly, our approach is also qualitative because it allows a direct structural examination of Abeta fibrils by fluorescence microscopy. In addition, this protocol can also be applied to the identification of substance(s) capable of inhibiting Abeta aggregation and further the assessment of the nature of such inhibition.
Chang
P
P
Institute of Pharmacology, College of Life Science, National Yang-Ming University, Shih-Pai, 11221, Taipei, Taiwan, ROC.
Su
Y
Y
D000544:Alzheimer Disease / Q000188:drug therapy; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000187:drug effects / Q000494:pharmacology; D001671:Biochemistry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000494:pharmacology; D013050:Spectrometry, Fluorescence
D016229:Amyloid beta-Peptides; D005456:Fluorescent Dyes; D010446:Peptide Fragments; C075222:amyloid beta-protein (1-42); D016650:Fluorescein-5-isothiocyanate