Hi all,
I am optimising ThT assay protocol for a-syn aggregation. Even if I perform 5 replicates, the graphs are having different lag times for the same sample. I am not too sure if there is a better way to ensure reproducibility between replicates?
Currently I have tried with 100uM and 20uM of wild type monomeric alpha synuclein protein shaking at 800cpm, with an without addition of NaCl as well. The ThT concentration I use in the final solution is 20uM.
Kindly refer to the image attached for 20uM monomer with 100mM NaCl (as referenced from literature). Kindly ignore the timestamp, as these were all transferred from a different plate reader (so t=0 is actually after around 48 hours of shaking elapsed).
Or is it ok to just take the average of these curves? This does not sound right to me as they all have different lag times and it would not be fair to just take the average of them.
Hope someone can advise!
Thanks and Regards,
Mathangi
One point I forgot to include, I used a 384-well plate, 50uL volume per well. As in the image attached with this comment, I started filling my well from row 2 of the plate, But I forgot to add PBS/water to the surrounding wells to tackle the issue of evaporation. I noticed that my first and last wells (denoted by arrows) have been showing decrease in the lag phase as opposed to the wells in between. Not sure if this might a reason.
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