Thioflavin T fluorescence assay

Commercially available Thioflavin T (ThT) was purchased from Sigma Aldrich and a stock solution of concentration 2,5 mM in Mili-Q water was prepared and stored the solution at −18 °C. Freshly received stock samples of Aβ 1–40 WT, H6A, H13A, and H14A in 10 mM CH₃COONH₄ (pH 7.4, when necessary the pH was adjusted with ammonia) were prepared to perform the fluorescence study, appropriate volume of peptide sample was taken out from the stock solution and was mixed with 10 μL of ThT solution (2,5 mM); to accomplish final peptide and ThT concentration of 100 μM, final volume was made up to 250 μL in 10 mM CH₃COONH₄. For ThT fluorescence assay, emission was measured from 450 nm to 650 nm and excitation at 440 nm, using a slit of 1 nm on a FP-6500 spectrofluorometer instrument (Jasco, Tokyo, Japan). The content of the cuvettes was pipetted up and down before every measurement. The fluorescence was measured at various time points, up to 72 h after the final SpeedVac purification step. From the machine text file was taken and graphs were plotted using OriginPro software (version 8, OriginLab Corporation, Massachusetts, USA).