Hello,
I am using ThS fluorescence as an indicator for tau aggregation in kinetics assays. This has been working really well, but I have now started to use the same assay to investigate the effect of a tau-aggregation inhibitor, which is blue when oxidised and clear when reduced. The inhibitor quenches the signal in my assays, and it seems that the signal is quenched significantly less when the inhibitor is reduced and in its clear form.
This has lead me to optimising my assay to minimise oxidation in my assay, using DTT in the sample, preparing the assay in a glove box with nitrogen and using lower amounts of the inhibitor. However, I am still getting quenching of the ThS signal.
Do you know if there are any other amyloid-detecting dyes that will not be quenched? Or, any more ideas to minimise oxidation in the reaction and keeping the sample clear?
Thank you!
First, record the absorption spectrum of your inhibitor in oxidised and reduced forms. If it's overlapping with ThS emission spectrum, you are going to have quenching.
You can try alternate amyloid-detecting dyes such as Congo red. If that doesn't work, labelling tau with another fluorophore might help. We have had success with introducing a tryptophan residue in tau and successfully used the tryptophan fluorescence to monitor aggregation kinetics.
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