Shift of ThT excitation maximum (from 385 nm to 450 nm) and its emission maximum (from 445 nm to 482 nm) occurs only upon binding to amyloid fibrils ( doi:10.1016/j.bbapap.2010.04.001). And we do not see the formation of amyloid fibirls in the lag phase, right? Then why does the shift occurs at such early "lag phase" stage?
ThT study is very confusing as I feel. There we need to see the emission of free and bound. They both have different excitation and emission. Bound one gives emission around 480 nm if you excite at 440 nm. The shift occurs due to binding. You can just test it. But here another issue will be the slit width. If it is lower, you will find a structural emission but in higher slit (5/10) you will find a clear single emission peak. These are just my observation. Recently we published an article where we have used ThT fluorescence to monitor fibrillation.
Uttam Pal
Hi
I get a ThT fluorescence signal of around 12-15 mAU even in the buffer. I have tried washing cuvette with 10% SDS and have tried HCl. However, the intensity in buffer comes the same every time.
I always filter the ThT working solution (100 uM) before use.
Devanshu Mehta - Amyloid fibers are likely bound to the surface of the cuvette from a previous experiment. SDS is often not sufficient to remove them. Try washing in aqua regia
It can also be due to formation of oligomeric/protofibrillar intermediates that can also bind ThT but with lower affinity compared to fibrils.
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