Electron microscopy of prefibrillar structures and amyloid fibrils

Odense University, Denmark.
Methods in Enzymology (Impact Factor: 2.09). 02/1999; 309:491-6. DOI: 10.1016/S0076-6879(99)09033-3
Source: PubMed


Several techniques, such as X-ray crystallography, light scattering, fluorescence spectrometry, size exclusion chromatography, atomic force microscopy, and transmission electron microscopy, have been employed in studies of structural intermediates of fibril formation and fibrillar assembly of amyloid proteins. Electron microscopy, with a resolution of approximately 2 nm, offers a useful technique for the ultrastructural characterization of preprotofilaments, protofilaments, and mature fibrils formed during in vitro fibrillogenesis. For contrast enhancement of specimens, negative staining is applied. This chapter outlines the methodology used in laboratories for electron microscopic examination of negatively stained prefibrillar structures and amyloid fibrils. The Aβ-peptide used influences the kinetics of the fibril formation. This chapter uses Aβ1–42 (Bachem, Bubendorf, Switzerland), which forms fibrils within a few hours of incubation at 37°. In order to decelerate the fibril formation enabling it to investigate early intermediates and prefibrillar structures, this chapter has performed the in vitro studies at low concentrations of Aβ1–42 (170 μg/ml), as the kinetics of fibril formation is highly concentration dependent.

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    • "The morphology of fibrils in water (pH 3.0) obtained by us is totally consistent with the previously obtained data [32]. "
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    ABSTRACT: The aim of this study was to investigate the process of amyloidogenesis of amyloid-β (Aβ)42 peptide, by means of fluorescence spectroscopy, electron microscopy, X-ray diffraction, and mass spectrometry. It has been repeatedly reported in the literature that the process of fibril formation by Aβ 42 peptide depends considerably not only upon the specific conditions (ionic conditions, pH, temperature, mixing, etc.), as well as the manufacturing route (synthetic or recombinant), but also on the methods of synthesis and purification. We have, for the first time, systematically analyzed samples of Aβ 42 peptide supplied by five different companies (Anaspec, Invitrogen, Enzo, Sigma-Aldrich, and SynthAssist) and obtained evidence of significant variability, including lot to lot variations. All studied samples formed amyloid-like fibrils at pH3-6, and the fibrils contained cross-β structures. Samples from Anaspec, Invitrogen, and Enzo formed one particular type of amyloid-like fibrils, while the samples from Sigma-Aldrich and SynthAssist formed another distinct type of fibrils. The observed polymorphism emphasizes the capacity of the Aβ 42 peptide to act as a prion agent with varying structural characteristics. The presented data have allowed us to propose a possible mechanism of formation of amyloid-like fibrils.
    Full-text · Article · Sep 2015 · Journal of Alzheimer's disease: JAD
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    • "The presence of disordered fibers (observed mainly in controlled pH) could be due to globular protein elements forming complex macro-aggregates. Similar fibers structures have been observed previously [78,79]. In addition, the differences in IBs size probably are due to differences in nucleation and IB growth properties during formation, as well the host proteins that interact with them. "
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    Full-text · Article · Sep 2014 · Microbial Cell Factories
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    • "All reagents used in peptide synthesis pentafluorophenol (Spectrochem Pvt. Ltd., Mumbai), N,N-dicyclohexyl carbodiimide (Sigma-Aldrich), Fmoc-AA (Novabiochem), HOBt (Sigma-Aldrich) and TFA (Sigma-Aldrich) were of the purest analytical grade. The Aβ peptide fibrils were formed by solubilizing the peptide in sterile double distilled (DD) water (1 mg/ml) and incubated for 24 h at 37 °C (Nielsen et al., 1999). The protofibrillar form thus obtained was used fresh for the present study. "
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