ThT is assayed by fluorescence intensity measurement. Sometimes protein aggregation left no much soluble/bound ThT. Unless the signal is strong, otherwise sedimentation of aggregation leads to lower/insiginifant ThT signal compared to blank.

Your question makes me think you did a UV/Vis-Measurement. The dye in both states absorbs the light approximately similarly well, its the internal conversation rate which is greatly inhanced in the bound state since the rotation in the molecule is prohibited. In simpler words: the energy of the dye is very quickly lost so its less likely to be converted into a photon. Therefore the quantum yield for the fluorescence is reduced, but the likelihood of the dye to absorb a photon is exactly the same.

Continuing the Christpoh answer it is needed to know which proteins is there (5 mg of what?) and what technique are you using; UV/VIS or fluorescence and in where (96-well plates, individual cuvettes, UV transparent)?

For expanding the proper answer more details are needed.

Dear Shrikant,

I am a bit confused with your question. ThT assay to monitor aggregation, relies in its fluorescence properties. When bound to beta-sheet structures, it shifts its fluorescence and when excited at 450nm, it starts emiting at 482nm. Therefore, with aggregation, the beta-sheet structure increases, and fluorescence should follow this trend.

For the case of synuclein, I recommend you to decrease its concentration. Usually people assay its aggregation kinetics in a concentration of 5 to 10 times less the one you are using. It gives you much more reproducible data.

You could also check one of our latest papers to see some aggregation kinetics by ThT assay.

Article Glycation potentiates α-synuclein-associated neurodegenerati...

Hope this helps.

Good luck!!