title: Thioflavin T as an amyloid dye: fibril quantification, optimal concentration and effect on aggregation.
abstract: Formation of amyloid fibrils underlies a wide range of human disorders, including Alzheimer's and prion diseases. The amyloid fibrils can be readily detected thanks to thioflavin T (ThT), a small molecule that gives strong fluorescence upon binding to amyloids. Using the amyloid fibrils of Aβ40 and Aβ42 involved in Alzheimer's disease, and of yeast prion protein Ure2, here we study three aspects of ThT binding to amyloids: quantification of amyloid fibrils using ThT, the optimal ThT concentration for monitoring amyloid formation and the effect of ThT on aggregation kinetics. We show that ThT fluorescence correlates linearly with amyloid concentration over ThT concentrations ranging from 0.2 to 500 µM. At a given amyloid concentration, the plot of ThT fluorescence versus ThT concentration exhibits a bell-shaped curve. The maximal fluorescence signal depends mostly on the total ThT concentration, rather than amyloid to ThT ratio. For the three proteins investigated, the maximal fluorescence is observed at ThT concentrations of 20-50 µM. Aggregation kinetics experiments in the presence of different ThT concentrations show that ThT has little effect on aggregation at concentrations of 20 µM or lower. ThT at concentrations of 50 µM or more could affect the shape of the aggregation curves, but this effect is protein-dependent and not universal.
authors.affiliation: Department of Neurology, Brain Research Institute, Molecular Biology Institute , University of California , Los Angeles, CA 90095 , USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Xue
authors.affiliation: Department of Neurology, Brain Research Institute, Molecular Biology Institute , University of California , Los Angeles, CA 90095 , USA.
authors.forename: Tiffany Yuwen
authors.initials: TY
authors.lastname: Lin
authors.affiliation: Department of Neurology, Brain Research Institute, Molecular Biology Institute , University of California , Los Angeles, CA 90095 , USA.
authors.forename: Dennis
authors.initials: D
authors.lastname: Chang
authors.affiliation: Department of Neurology, Brain Research Institute, Molecular Biology Institute , University of California , Los Angeles, CA 90095 , USA.
authors.forename: Zhefeng
authors.identifier: 0000-0003-1992-7255
authors.initials: Z
authors.lastname: Guo
keywords: Alzheimer's disease; Aβ; fibrillization kinetics; yeast prion