Figure 2

Spectrofluorimetric analysis of purified ShadowY. (a) Normalized excitation spectra of mEGFP, Clover, sREACh, and ShadowY. (b) The emission spectra of mEGFP, Clover, sREACh, and ShadowY excited at 450 nm. For all the samples, optical density at 450 nm is adjusted to the same value. (right) An enlarged view of left panel. (c) Two-photon excitation spectra of 5 µM mEGFP, 5 µM Clover, 5 µM Clover_T153MF223R, 30 µM sREACh, 100 µM ShadowG, 100 µM ShadowY. The measured fluorescence intensities were divided by the respective protein concentrations, and the peak intensity of Clover was normalized to 100. (right) An enlarged view of left panel. (d) Fluorescence lifetime curves of the fluorescent proteins subjected to 2-photon excitation at 920 nm. Measured fluorescence intensity was divided by the respective protein concentration. (inset) Normalized fluorescence lifetime curves. (e,f) Fluorescence recovery of sREACh and ShadowY from a denatured (e) or reduced state (f). The respective fluorescent protein was excited at 517 nm with 5 nm bandwidth, and its fluorescence recovery was monitored at 531 nm with 5 nm bandwidth. Three independent experiments were averaged (the data are shown as mean ± SEM).