title: Fluorescent filter-trap assay for amyloid fibril formation kinetics in complex solutions.
abstract: Amyloid fibrils are the most distinct components of the plaques associated with various neurodegenerative diseases. Kinetic studies of amyloid fibril formation shed light on the microscopic mechanisms that underlie this process as well as the contributions of internal and external factors to the interplay between different mechanistic steps. Thioflavin T is a widely used noncovalent fluorescent probe for monitoring amyloid fibril formation; however, it may suffer from limitations due to the unspecific interactions between the dye and the additives. Here, we present the results of a filter-trap assay combined with the detection of fluorescently labeled amyloid β (Aβ) peptide. The filter-trap assay separates formed aggregates based on size, and the fluorescent label attached to Aβ allows for their detection. The times of half completion of the process (t1/2) obtained by the filter-trap assay are comparable to values from the ThT assay. High concentrations of human serum albumin (HSA) and carboxyl-modified polystyrene nanoparticles lead to an elevated ThT signal, masking a possible fibril formation event. The filter-trap assay allows fibril formation to be studied in the presence of those substances and shows that Aβ fibril formation is kinetically inhibited by HSA and that the amount of fibrils formed are reduced. In contrast, nanoparticles exhibit a dual-behavior governed by their concentration.
authors.affiliation: Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, P.O. Box 124, SE 221 00, Lund, Sweden.
authors.forename: Irem
authors.initials: I
authors.lastname: Nasir
authors.affiliation: Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, P.O. Box 124, SE 221 00, Lund, Sweden.
authors.forename: Sara
authors.initials: S
authors.lastname: Linse
authors.affiliation: Department of Biochemistry and Structural Biology, Center for Molecular Protein Science, Lund University, P.O. Box 124, SE 221 00, Lund, Sweden.
authors.forename: Celia
authors.initials: C
authors.lastname: Cabaleiro-Lago
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D016229:Amyloid beta-Peptides / Q000737:chemistry; D052160:Benzothiazoles; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004926:Escherichia coli; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles; D010446:Peptide Fragments / Q000737:chemistry; D011137:Polystyrenes; D055503:Protein Multimerization; D012709:Serum Albumin / Q000737:chemistry; D012996:Solutions; D013844:Thiazoles
chemical_list: D000682:Amyloid; D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011137:Polystyrenes; D012709:Serum Albumin; D012996:Solutions; D013844:Thiazoles; C075222:amyloid beta-protein (1-42); C009462:thioflavin T
keywords: Alzheimer’s disease; Amyloid; aggregation; filter-trap; kinetics; site-specific fluorophore labeling