Thioflavin T (ThT) spectroscopic assay

Principle:

Thioflavin T (ThT) is a benzothiazole salt obtained by the methylation ofdehydrothiotoluidine with methanol in the presence of hydrochloric acid. ThT is used as a dye to visualize and quantify the presence or fibrilization of misfolded protein aggregates, or amyloid, both in vitro and in vivo (e.g.,plaques composed of amyloid beta found in the brains of Alzheimer's disease patients; plaques of PrP fibrils found in brains of CGD patients).

The Thioflavin T (ThT) Assay measures changes of influorescence intensity of ThT upon binding to amyloid fibrils. The enhanced fluorescence can be observed by fluorescence microscopy or by fluorescent spectroscopy. The spectroscopic assay is commonly used to monitor fibrilization over time, but the assay is not strictly quantitative and differences in binding have been observed for samples after lyophilization.

Protocol:

1. Prepare a ThT stock solution by adding 8mg ThT to 10mL phosphate buffer (10mM phosphate, 150mM NaCl, pH 7.0) and filter through a 0.2μm syringe filter. This stock solution should be stored in the dark and is stable for about one week.

2. Dilute the stock solution into the phosphate buffer (1mL ThT stock to 50mL buffer) on the day of analysis to generate the working solution.

3. Measure the fluorescence intensity of 1mL working solution by excitation at 440 nm (slitwidth 5 nm) and emission 482 nm (slitwidth 10 nm), averaging over 60 s.

4. Add an aliquot of untreated protein solution (5–10μL) to the cuvette, stir for 1 min, and measure the intensity over 60 s. This serves as the control sample.

5. Repeat steps 3–4 with 5–10µL of the aggregated protein solution. A measured intensity above the control sample is indicative of amyloid fibrils.

6. Note: this ThT assay protocol is for testing in a cuvette,and is valid if you use multi-scan spectrometer that requires multi-well plates. (nurc, black)

Tips:

1. Final ThT value:

Due to difference of spectrometers one may use, final ThT values differ. Generally, for multiple scanning spectrometers like products of Thermo, Omega, their data represents relative influorescence value but the process saves time. For influorescence spectrometers like products of Hitachi, their data represents absolute influorescence value and the values are more acurate, but samples need to be tested one by one in a cuvette.

2. Incubation:

For protein fibrilar formation, it normally needs a certain length of time. Different proteins require different temperature and time for fibrilization incubation. Most spectrometers have functions of setting incubation temperature and time. Make sure you set the appropriate temperature and time period you need for your proteins. Moreover, some proteins need continuous shaking.

3. For multiple scanning spectrometer, the multi-well plates need to be black and intransparent.