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      Thioflavin T as an amyloid dye: fibril quantification, optimal concentration and effect on aggregation

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          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.

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          Most cited references 36

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          Molecular mechanism of Thioflavin-T binding to amyloid fibrils.

          Intense efforts to detect, diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of amyloid-specific molecular probes. Since its first description in 1959, the fluorescent dye Thioflavin-T (ThT) has become among the most widely used "gold standards" for selectively staining and identifying amyloid fibrils both in vivo and in vitro. The large enhancement of its fluorescence emission upon binding to fibrils makes ThT a particularly powerful and convenient tool. Despite its widespread use in clinical and basic science applications, the molecular mechanism for the ability of ThT to recognize diverse types of amyloid fibrils and for the dye's characteristic fluorescence has only begun to be elucidated. Here, we review recent progress in the understanding of ThT-fibril interactions at an atomic resolution. These studies have yielded important insights into amyloid structures and the processes of fibril formation, and they also offer guidance for designing the next generation of amyloid assembly diagnostics, inhibitors, and therapeutics. Copyright (c) 2010 Elsevier B.V. All rights reserved.
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            Spectroscopic determination of tryptophan and tyrosine in proteins.

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              Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1.

              We used a fluorometric method to examine amyloid fibrils, in vitro. These fibrils in the case of both murine senile and secondary amyloidosis were purified to apparent homogeneity from the water-suspended fraction of the liver of senescence-accelerated mouse, using sucrose density ultracentrifugation, and then the following assays were performed. In the absence of amyloid fibrils, thioflavine T fluoresced faintly at the excitation and emission maxima of 350 and 438 nm, respectively. In the presence of amyloid fibrils, thioflavine T fluoresced brightly at the excitation and emission maxima of 450 and 482 nm, respectively, and the fluorescence change was linear from 0 to 2.0 micrograms/ml amyloid fibrils. This fluorescence was maximal around pH 9.0. Fluorescence intensity in the presence of a constant amount of amyloid fibrils reached a plateau with increase in the thioflavine T concentration. Normal high density lipoproteins which contain apo A-II, the precursor of amyloid fibrils in murine senile amyloidosis, and acute phase high density lipoproteins which contain serum amyloid protein A, the precursor of amyloid fibrils in secondary amyloidosis, showed little fluorescence. The fluorescence was considerably diminished when structure of the amyloid fibrils was disrupted by guanidine-HCl treatment. This method will be useful for the determination of amyloid fibrils in vitro.
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                Author and article information

                Journal
                R Soc Open Sci
                R Soc Open Sci
                RSOS
                royopensci
                Royal Society Open Science
                The Royal Society Publishing
                2054-5703
                January 2017
                4 January 2017
                4 January 2017
                : 4
                : 1
                Affiliations
                Department of Neurology, Brain Research Institute, Molecular Biology Institute, University of California , Los Angeles, CA 90095, USA
                Author notes
                Author for correspondence: Zhefeng Guo e-mail: zhefeng@ 123456ucla.edu
                Article
                rsos160696
                10.1098/rsos.160696
                5319338
                © 2017 The Authors.

                Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

                Product
                Funding
                Funded by: National Institute of General Medical Sciences http://dx.doi.org/10.13039/100000057
                Award ID: R01GM110448
                Categories
                1001
                15
                Biochemistry and Biophysics
                Research Article
                Custom metadata
                January, 2017

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