Binding stability of Ts1-S14R-Bodipy characterized by LRET using DI-3GLBT-R(-5). (a) Time constant of DA signals (red squares) are accelerated as the concentration of Ts1-S14R-Bodipy is increased, whereas that of SE signals (green circles) show a fixed value, around 1.4 ms (0 µM and 0.25 µM: n = 8; 0.5 µM: n = 6; 1 µM: n = 6; and, 2 µM: n = 4). The apparent dose-response curve of DA signals indicate that the proportion of Ts1-S14R-Bodipy binding to DI-3GLBT-R(-5) reach 50% at around 0.5 µM and reach 100% at around 1 µM. (b) The dissociation rate of Ts1-S14R-Bodipy as a function of time in toxin-free solution is very slow. We evaluated the binding fraction of Ts1-S14R-Bodipy by the amplitude of SE signals (n = 8). At 40 minutes after pretreatment by Ts1-S14R-Bodipy, 85% of the molecules are still bound. (c) Representative raw traces of SE signal at Time 0 min (green) and at Time 40 min (orange) in B.