Characterization of T-Sapphire A: Refolding of Sapphire and T-Sapphire from their denatured states prepared by incubation in denaturation buffer (8M urea, 1 mM DTT) at 95°C for 2 min. Recovery of fluorescence (emission at 511 nm and excitation at 399 nm) was initiated by 100fold dilution into renaturation buffer (35 mM Kcl, 2 mM MgCl2, 1 mM DTT, 50 mM Tris pH 7.5). B: pH-titration of fluorescence of T-Sapphire. Excitation was at 399 nm and emission at 511 nm. D: Absorbance of equal amounts of T-Sapphire protein at pH 4, 5, 6 and 7. No significant differences were detected.