Hello, I already tested by NMR the interaction of an antibody-amyloid beta and now I want to test the influence in amyloid beta 1-42 aggregation kinetics of the presence of an antibody in solution. Due to the availability of equipments my first option is tu use ThT assay.
My main doubt is if the ThT will interact with the antibody (mouse IgG1) in solution and give me masked results.
Of course, I will use controls for assessing that hypothetical influence, but if that instruction will be enough for making useless the assay, I will try another option.
Thank you very much!
Why do not you perform both congo red assay and ThT assay and compare the outcome of both. Also you might need to investigate the interaction of ThT and the antibody in absence of amyloid beta 1-42 as a control experiment
Hi Adrián, if ThT is giving you trouble, then there are other dyes you can use.
Article Common Benzothiazole and Benzoxazole Fluorescent DNA Interca...
Another problem I am seeing in my experiment is not only that antibody could interact with ThT and give wrong measurements, but compete with ThT for binding the amyloid peptides/aggregates. I saw by NMR that my antibody could bind amyloid beta in the same residues that ThT...
ThT is proposed to bind to amyloid fibrils in a specific benzothiazole binding site. Therefore, I expect that using other benzothiazole based dyes or isosteres of benzothiazoles will result in the same issue you are observing now. I strongly recommend you compare your results to another non-benzothiazole based dye which potentially would bind to another site in the amyloid fibrils
Thank you all for the advices! I will read a bit more about my options and I´ll let you know my decision and results.
;)
Hi Ben, thank you.
Anyway, I am trying to monitor aggregation using ThT and I am having outrageous results. Final concentrations and conditions:
- Abeta 1-42 (pretreated with NH4OH and resuspended in NaOH (no sanitation, no vortexing): 56 µM
- ThT: 0.2 mM (in PBS)
- PBS 10 mM till 100 µL
- Greiner black flat bottom plates
Everything at 4ºC till plating and start measures (440 nm exc and 484 nm emission) each 5 min, 15 sec shaking (6 mm amplitude) before measurement, 37ºC. Equipment (tecan infinite 200) gain setting at 140.
Fluorescence values don´t increase but even go down 5-10 min from assay start and stabilize. I repeated with 3 different Abeta aliquots and I see the same. Maybe my aliquots are a bit old... but in ELISA still worked fine.
Any suggestion? Thanks in advance!