As per literature, the excitation wavelength of alpha-synuclein is around 440nm (and the emission wavelength 485nm). In our setup we have a fixed concentration of the fibrils and thioflavin T concentration ranging from 1nM to 300uM in order to try and obtain a dose-response curve.
During the first round of experiments we only had a 460nm excitation filter available and had the correct emission filter. We were not able to get any kind of dose-response. This time round we had a 450nm excitation filter, and got a dose-response curve, but the curve dipped down at the higher concentrations of Thioflavin T (image is attached). Is this normal? Is this due to micelle formation that is supposed to occur at higher concentrations of Thioflavin T? We really don't need to use concentrations above 10uM and could just stop the curve there. Or should we be trying to obtain a 440nm excitation filter?
Dear Nisha,
starting with your main question. Excitation wavelength can be crucial for obtaining the signal. From my experience at 460 nm you are at around 10 % of maximum intensity of THt while at 450 nm it is reaching 50-60 %. Of course it is highly dependent on the buffer you are using (some components may shift the maximum excitation wavelength) and bandwidth of the filter used (larger bandwidth give better signal, but it is also less specific).
About the drop in dose response, it is hard to say if it is normal. I do not know anyone who used such high concentrations of ThT for amyloid studies. However, you are right that it might be connected with micelle formation (cmc for ThT is around 4 um, http://www.ncbi.nlm.nih.gov/pubmed/16125973). Therefore, when using high ThT concentration the micelle formation might be a more favourable process and therefore there is less ThT molecules left to bind fibrils.
And finally, buying 440 nm filter will increase your signal, but it will not change the fact that fluorescence signal is dropping on your dose response curve.
I hope this answer will be helpful.
Best regards
http://www.ncbi.nlm.nih.gov/pubmed/16125973
Thank you very much for your reply Wojciech, it was indeed helpful.
I think we will stop the curve at 30uM and go ahead to the next step (blocking the fluorescence with another compound) with the 450nm filter and then decide on getting the 440nm filter based on the outcome.
Since posting the question, I have come across this study where they found that the fluorescence dropped at concentrations of Thioflavin T above 50uM, which is similar to what we found.
http://www.phys.ufl.edu/REU/2006/reports/riggs.pdf
Regards,
Nisha
Another potential explanation for the decreased fluorescence at higher concentrations is the inner filter effect, in which the absorbance of the excitation and/or emission light by the compound itself reduces the fluorescence intensity. This effect can be reduced, if necessary, by shortening the pathlength by using a narrower cuvette or reducing the volume in an assay plate, or by changing to an off-peak excitation and/or emission wavelength.
Since you have a very large signal already with the 450 nm filter, I don't think it is necessary to buy a 440 nm filter. However, you haven't shown the curve in the absence of alpha-synuclein. It may be that the ratio of fluorescence + alpha-synuclein to fluorescence - alpha-synuclein would be higher with the optimal filter.
Dear Adam,
Thank you. Is the curve in the absence of alpha-synuclein that you mentioned something I should be doing each time or just once to check? I have only checked this at one concentration of Thioflavin-T (3uM) and have not made a whole curve. At this concentration, the fluorescence was about hundred times higher in the presence of alpha-synuclein.
I would subtract the curve of fluorescence v. Thioflavin T concentration in the absence of alpha-synuclein from the curve in its presence in order to measure the synuclein-dependent Thioflavin fluorescence response. However, if the fluorescence in the absence of alpha-synuclein is negligibly small (
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