Thioflavine T Binding Assay
 

 

THIOFLAVINE T BINDING IN 96-WELL PLATES

 

This protocol is to be used to measure amyloid fibrils (or aggregates) at concentrations of 0.1-0.4 mg/ml. If much higher or much lower concentration is used, it might need readjustment.

 

Thioflavine T Solution: 10 µM in 50 mM Tris, 100 mM KCl, 1mM EDTA, pH 8.0

Procedure:

-Mix fibrils or solution to analyze 5 sec in vortex to have homogeneous solution

-Transfer 25 µL into 3 wells of a black or white 96 well plate (not transparent), samples and blanks.

-Add 175 µL of Thioflavine T solution to each well

-Measure Fluorescence:

With Safire2 we have the following conditions:

                            EXCITATION 440 NM; EMISSION 482 NM (bandwidth 5nm)

 

Thioflavine T (ThT) MW: 318.9

-Prepare a concentrated stock of 10 mM (1000X) in Tris Buffer: 31.9 mg ThT in 10 mL Tris Buffer

-Prepare working solution by diluting the stock: 50 ml Tris Buffer + 50 ul 1000X stock

 

Tris Buffer, pH 8.0 (500 mL)

25 ml               1M Tris, pH 8.0

25 ml               2M KCl

1 ml                 0.5M EDTA

                        Water till 500 mL