Dear all,
I am currently working on amyloid beta aggregation. In ThT monitered aggregation assay, I have already obeserved an increase in ThT signal, indicating the aggregation of amyloid beta. However, after a certain time the fluorescence signal changes dramatically in all wells (as shown in figure). The concentration of A-beta I used in ThT assay is 5 uM and the concentration of ThT is 20 uM and the fluorescence signal was monitered for 48 hours. ThT emission was measured at 5min intervals with 3 sec orbital shaking before measurement. Besides, I sealed the 96-well plate before assay so I do not think it is the problem of evaporation. How can I optimized my experiment to get a smoother curve?
Thanks!
Hello!
As far as I know, the curves become noisy because of the fibrils, which start to be a big aggregate, and they sink down and go back to the solution in an uncontrolled way.
Have you tried to stir your solution more vigorously or simply for more seconds?
I wish you good luck with your future attempts.
Another possible contribution to the spikes is the formation of air bubbles in the wells.
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