About the Protocol
ThioflavinT (ThT) is a fluorescence dye that fluoresce upon binding to amyloid aggregates. In our lab we use this property of ThT to study aggregation kinetics of amyloidogenic proteins and peptides. Typically, the protein of interest is kept under aggregating conditions and at definite interval of time aliquots are taken and extent of aggreagtion is measured by ThT fluorescence.
Reagents ThioflavinT Sigma)
10 mM Phosphate buffer (pH 7.4) or 10mM Glycine-NaOH (pH 9.8)
Preparing ThT stock solution
1.Weigh out .... mg of ThT and dissolve it in ... ml of MiliQ water.
2.Take 10μl of this solution and add to 990μl of MiliQ water and take the absorbance at 412nm (ε412 = 36,000 M-1 cm-1)
3.Calculate the concentration of the stock using Beer-Lambert's law: A= ε×C×L (C is the concentration in molar unit and L is the path length of the cuvette)
4.Keep the stock at 4°C wrapped in aluminium foil.
[Notes from an experienced user:]
1.An attempt to prepare a 5mM stock solution (by weight) ends up in 3mM solution by absorbance
2.The stock may be kept at 4°C for 3 months. Its advisable to prepare as fresh stock after 3 months or to recheck the the concentration by absorbance.
Measuring Tht fluorescence
1. Prepare a substock of 20μM ThT from the 3mM stock in phosphate buffer. For some proteins like α-synuclein Glycine NaOH (pH 9.8) works better. Keep in a 15ml falcon tube.
2. Wrap the falcon with aluminium foil to protect from light. Do not use this solution after 24h.
3. Take 300μl of this solution in a cuvette and add to it 50μl of the protein solution whose aggregation is being studied.
4. Incubate for 5 min in dark.
5. At the end of 5 min take the fluorescence (λex: 450nm; λem: 484 nm) with slit width 5nm and 10nm for excitation and emission respectively