I am trying to perform thioflavin -T aggregation kinetics with 20 micromolar as well as 50 micromolar concentration of amyloid beta.
I am not getting the regular aggregation kinetics curve when i measure fluorescence intensity at 440nm ex and 490nm em. I think this might be due to presence of oligomers of high molecular weight even after NaOH treatment and sonication for 1 minute in ultrasonication bath. I wish to try ultracentrfugation and then getting monomers for performing aggregation assay. Please let me know how to proceed with ultracentrifugation( speed and time). Also suggestions are welome if i could get good results for my thioflavin-T aggregation assay.
Hi, I know this is a little old but I spent a decent amount of time trying to figure out issues with ThT kinetics on IAPP (amylin) and I tested my ideas on Abeta40 also. I'll link the article we did on this for you, it's quite short but briefly we found that due to the nature of microplate readers, the measurement frequency can drastically alter kinetics. So both how often and how many wells you measure can act as a source of agitation and accelerate kinetics. I hope this helps.
Article Thioflavin T fluorescence to analyse amyloid formation kinet...
Hi Matthew,
I tried your protocol. It seems that my peptide is not aggregating. Please suggest me what can go wrong while putting out for aggregation of such a recombinant peptide with high aggregation propensity.
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