Thioflavin T (ThT) fluorescence kinetics

Thioflavin T (ThT) binding assay was carried out to quantify OVA amyloid formation and also to examine the impact of CsA on this process. ThT fluorescence assay has been widely used to detect cross -β sheet structure of amyloids. The growth of amyloid formation is accompanied by building of a cross-β sheet structure, that readily binds with ThT and eventually ensues in a significant rise in fluorescence intensity.

Briefly, OVA aggregates (100 μg) obtained at various time points were incubated with 30 μM ThT solution (30 μl of 1 mM ThT stock solution) at room temperature. ThT specific amyloid fluorescence was measured on Hitachi F-4500 fluorescence spectrophotometer. The sample was excited at 450 nm and spectra were recorded at wavelength range of 450 nm to 600 nm. The excitation and emission slit widths were fixed at 5 nm and 10 nm respectively.