Hi Karen, ThT only binds to preformed amyloid complexes. I don't know about p53 amyloids, but maybe you should try out the technique on Ab 1-42 to see if it works.

There are also other fluorophores you can use to detect amyloids. 

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Hi Karen,

A couple thoughts here. First, in contrast to Steingrimur's experience, I have had success adding ThT to solutions of monomeric alpha Synuclein prior and had it report fibrillization in realtime. Some worry about singlet oxygen formation from ThT and recommend against it, and I decided to helium sparge solutions prior to be on the safe side (i.e. avoid reviewer comments!). Honestly I don't know that it matters.

Second, a lot of amyloid protocols use some sort of agitation to initiate fibrillization. The air-water interface makes a hydrophobic-hydrophilic interface that can unfold and orient at least some protein/peptides into an amyloid-favorable conformation.

Third, this may be instrumental. What kind of fluorometer do you have? If it's photon counting, you can saturate the counting board with too many photons and it spuriously reads low. Increased fluorescence will then read as decreased signal. The thing to try is to decrease the bandpass on excitation and/or emission to get all measured signals below the threshold. Check with your instrument manufacturer to see what the max photon rate is for detector linearity. Alternatively you can keep the bandpasses the same and use a light-reducing filter (e.g. neutral density filter) on excitation to reduce the light throughput.

Good luck!

Hi Karen,

It might take a while to establish suitable parameters for aggregation assays. Please consider the following things might be relevant

• aggregation time (6-72 hours for a lot of proteins)
• protein concentration (1-100 µM) compare to signal/ concentration relation in Fig 6B of linked article)
• use not more than equimolar ThT
• optimized excitation (420-440nm) and emission (480-520nm) Some proteins prefer different parameters)
• agitation like shaking every 15 min before aggregation
• pH (could be between pH 2-10)
• salt concentrations NaCl, CaCl2, KCl
• mild denaturation with urea
• reducing environment (e.g.DTT)
• if you have access to a plate reader try several buffer conditions at once, for my first assay I just grabbed several buffers we had available in the lab
• check the gain settings in the fluorescence reader

FYI: You can mix ThT solution in advanced, filter it through a 0.2 µm syringe filter the solution a 500µM solution can be stored at 4°C in the fridge for several days (please check also your ThT dilution was correct.)

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Hi Karen,

I met the same problem that ThT signal decreases over time (both with and without fibrils). I wonder if you have solved this problem and how you solved this. Thank you.