Thioflavin t

Manufactured by Agilent Technologies

Thioflavin-T is a fluorescent dye used for the detection and quantification of amyloid fibrils. It is a core component in the analysis of protein aggregation and misfolding. Thioflavin-T binds to the cross-beta sheet structure of amyloid fibrils, resulting in an enhanced fluorescence signal that can be measured spectrophotometrically.

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Lab products found in correlation

Thioflavin t, by Abcam (2 mentions) Kaleidagraph, by Synergy Software (2 mentions) Gen5 microplate reader, by Agilent Technologies (2 mentions) Thioflavin t, by Merck Group (2 mentions) Synergy mx, by Agilent Technologies (1 mentions) Jem 2100f transmission electron microscope, by JEOL (1 mentions) Flx800, by Agilent Technologies (1 mentions)

4 protocols using thioflavin t

Thioflavin-T Kinetics of TDP-43 Aggregation

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Myo-granules were isolated from myotubes and diluted in phosphate buffered saline. Three separate biological replicates were performed constituting purification from three separate myotube cultures. 25 uM Thioflavin-T (Abcam) was added to recombinant 15uM HIS-SUMO-TDP-43, myo-granule, or myo-granule plus recombinant 15uM HIS-SUMO-TDP-43. Surface denaturation was performed with continuous shaking at 37°C with Thioflavin-T incorporation monitored every 10 minutes at 495 nm after excitation at 438 nm on a Gen5 microplate reader (BioTek). Raw fluorescence values obtained for experimental conditions were background subtracted and plotted as a function of time. The resulting curves were fit to following single exponential rate equation (Equation 1) using Kaleidagraph (Synergy Software).

- A * e^{(- k o b s \times t)} + B .

where A is amplitude, k_obs (min⁻¹) is single exponential rate constant, and B represents maximal amount of fluorescence detected.

Vogler T.O., Wheeler J.R., Nguyen E.D., Hughes M.P., Britson K.A., Lester E., Rao B., Dalla Betta N., Whitney O.N., Ewachiw T.E., Gomes E., Shorter J., Lloyd T.E., Eisenberg D.S., Taylor J.P., Johnson A.M., Olwin B.B, & Parker R. (2018). TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle. Nature, 563(7732), 508-513.

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Thioflavin-T Kinetics of TDP-43 Aggregation

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- A * e^{(- k o b s \times t)} + B .

where A is amplitude, k_obs (min⁻¹) is single exponential rate constant, and B represents maximal amount of fluorescence detected.

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Thioflavin T Fluorescence Assay for Protein Aggregation

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Samples for Thioflavin T fluorescence analyses were prepared by combining 74 μl of each sample, 100 μl of 40% acetonitrile, and 1 μl of 10 mM Thioflavin T (CAS# 2390-54-7 purchased from Sigma-Aldrich) and measured in the wells of a 96 well plate (Microplate, 96 well, PS, F-bottom, μCLEAR, black, med. binding, Greiner Bio-one). Thioflavin T fluorescence was determined with a BioTek Synergy Mx plate reader (Serial# 250843) every 15 min for up to 24 h, with short shaking before each read. The excitation wavelength was 444 nm and fluorescence was measured at 484 nm. A well containing 1 μl 10 mM ThT in 40% MeCN and 174 μl 40% MeCN was used as a baseline. The plate was held at 37°C for all 24 h.

Gordon-Kim C., Rha A., Poppitz G.A., Smith-Carpenter J., Luu R., Roberson A.B., Conklin R., Blake A, & Lynn D.G. (2022). Polyanion order controls liquid-to-solid phase transition in peptide/nucleic acid co-assembly. Frontiers in Molecular Biosciences, 9, 991728.

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Cryo-TEM analysis of α-synuclein aggregation

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SNCA-His proteins were prepared as previously described [75 (link)]. The purified SNCA-His protein (the final concentration, 20 μM) was continuously shaken in phosphate-buffered saline (PBS) at 37° C for 3 days. For examination of α-synuclein aggregates, the formed fibrils were placed on a 200-Mesh copper (holey-carbon) grid and immerged into liquid nitrogen. For cryo-TEM examination, samples were inserted into a CP3 cryoholder (Gatan, Inc., Pleasanton, CA, USA) and observed at 200 kV by JEM-2100F transmission electron microscope (JEOL, Tokyo, Japan). Images were captured by using a 1024 × 1024 CCD camera (Gatan). For α-synuclein aggregate quantification, thioflavin T (10 μM final concentration; Sigma-Aldrich), a dye exhibiting enhanced fluorescence upon binding to amyloid fibrils [76 (link)], was added to the aggregated α-synuclein (± trehalose, LM-021 or NC009-1; 1−10 μM) and incubated for 5 min at room temperature. Trehalose was included as a positive control for inhibiting α-synuclein amyloid fibril formation [75 (link)]. Dimethyl sulfoxide in 1% was used as a negative control. thioflavin T fluorescence intensity of samples was recorded by using a microplate reader (Bio-Tek FLx800), with a combination of excitation at 420/50 nm and emission at 485/20 filter.

Yang P.N., Chen W.L., Lee J.W., Lin C.H., Chen Y.R., Lin C.Y., Lin W., Yao C.F., Wu Y.R., Chang K.H., Chen C.M, & Lee-Chen G.J. (2023). Coumarin-chalcone hybrid LM-021 and indole derivative NC009-1 targeting inflammation and oxidative stress to protect BE(2)-M17 cells against α-synuclein toxicity. Aging (Albany NY), 15(16), 8061-8089.

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