Mtp 810

Kinetics of amyloid fibrillation at 37 ºC was monitored using 5 μM hIAPP in 20 mM HEPES buffer (pH 7.4) containing 5 μM ThT and 50 mM NaCl in the presence and absence of LUVs. Other effectors of hIAPP amyloid aggregation including Zn(II), EGCG, or 1:1 molar ratio of EGCG:Zn(II) were added to the hIAPP sample solution at the desired concentration. Three independent experimental measurements were carried out for each sample.
ThT-based monitoring was performed using a sealed 96-well microplate (Greiner-Bio-One, Tokyo, Japan). ThT fluorescence intensity was observed in a microplate reader (MTP-810, Corona Electric Co. Ibaraki, Japan) every 3 min following a 10 s orbital shaking (strong mode). The excitation and emission wavelengths were set at 450 and 490 nm, respectively. Kinetic parameters of amyloid formation were obtained using the following equation;[28 (link)]
$$\text{Y}=y_i+m_i\text{t}+\frac{y_f+m_{f}t}{1+\exp \left[-k\left(t-t_0\right)\right]}$$
where y_i + m_it and y_f + m_ft indicate initial and final baselines, respectively. k and t indicate the rate constant and elongation time, respectively. t₀ is the half-time when ThT fluorescence reaches 50% of the maximum intensity. The lag time was calculated with the relationship, lag time = t₀ - 2(1/k).