Kinase experiment:

Thioflavin T (ThT) is prepared by dissolving ~3 mg dry powder in 1 mL water. The solution is filtered through 0.22 μm syringe filters followed by measurement of the concentration by diluting the stock solution in ethanol and using an extinction coefficient of 26,620 M-1 cm-1 at 416 nm. The stock solution is stored at 4 ℃ covered with foil and used for up to a month to make assay solutions by diluting either in water or desired buffer[1]. Thioflavin T (ThT) fluorescence emission is measured with excitation at 450 nm and recording the spectrum between 465 and 565 nm with 5 nm slits using a FluoroMax 2 spectrofluorometer. The excitation spectra are collected by setting the emission wavelength to 482 nm and collecting the spectrum between 300 and 470 nm with 5 nm slit widths, and 1 s integration time and 1 nm interval. Emission spectra between 465 and 565 nm are collected upon excitation at 450 nm. Excitation and emission spectra in the presence of amyloid fibrils are measured with varying concentrations of Thioflavin T and 5 ng/mL amyloid (calculated based on starting protein concentration) before collecting the spectra[1].

Thioflavin T is a cationic Benzothiazole dye that shows enhanced fluorescence upon binding to amyloid in tissue sections.

Thioflavin T (ThT) is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, Thioflavin T is found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay (~10-20 μM). Specific conductivity changes are measured at varying concentration of Thioflavin T and the critical micellar concentration is calculated to be 4.0±0.5 μM. Changes in the fluorescence excitation and emission of Thioflavin T are also dependent on the micelle formation. The Thioflavin T micelles of 3 nm diameter are directly visualized using atomic force microscopy, and bound Thioflavin T micelles are observed along the fibril length for representative fibrils. Increasing concentration of Thioflavin T above the critical micellar concentration shows increased numbers of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles are disrupted at low pH as observed by atomic force microscopy and fluorescence enhancement upon binding of Thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3.The micelles of Thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission[1].

[1]. Khurana R, et al. Mechanism of thioflavin T binding to amyloid fibrils. J Struct Biol. 2005 Sep;151(3):229-38.