Fluorescence Spectra Characteristics
Fluorescence spectra were taken with a Varian Cary Eclipse Fluorescence Spectrophotometer. For quantum yield determination, absorbance spectra of proteins were taken with a Cary UV-Vis spectrophotometer. For extinction coefficient determination, native protein absorbance was measured with the spectrophotometer, and protein concentration was measured by the BCA method. The excitation spectra of GFPSpark® is 487nm，and the emission spectra is 509nm. The quantum yield of GFPSpark® is 0.62 and EGFP is 0.60.The extinction coefficient of GFPSpark® is 47000 and EGFP is 39000. The extinction coefficient of GFPSpark® is better than EGFP.
Refolding and Maturation Kinetics
Samples of fluorescent proteins were heated to 95 ℃ in denaturation solution (8M urea,1mM dithiothreitol) for 4 min. Refolding reactions were initiated following 100-fold dilution in the renaturation buffer (35mM KCl, 2mM MgCl2, 50mM Tris pH 7.5, 1mM dithiothreitol). In the maturation assay, 5mM freshly dissolved dithionite was added to the denaturation solution (Reid & Flynn, 1997). Owing to the instability of dithionite at high temperatures and to provide for complete chromophore reduction, the sample was cooled to 25 ℃ and the addition of 5mM dithionite followed by heating to 95 ℃ were repeated. Protein refolding and maturation were followed by measuring the recovery of fluorescence using the Varian Cary Eclipse Fluorescence Spectrophotometer, with the chamber temperature maintained at 25 ℃. The refolding half time of GFPSpark® is 270s and EGFP is 570s. The maturation half time of GFPSPrk is 1305s and EGFP is 1560s.The maturation of GFPSpark® is faster than EGFP.
The pH sensitivity of GFPSpark® was determined in a 96-well format by adding 100 µl of dilute GFPSpark® in a weakly buffered solution to 100 µl of strongly buffered pH solutions in triplicate (total 200 µl per well) for pHs from 3 to 12. The fluorescence of each well was measured. The pH value which Fluorescence is 50% of the optimum pH had been defined as pKa of Fluorescence protein . The pKa of GFPSpark® is 4.5.The pKa of EGFP is 5. The pH stability of GFPSpark® is better than EGFP.
Laser scanning confocal microscopy (LSCM) photobleaching experiments were conducted with the GFPSpark®. HeLa cells were transfected with the expression vector for at least 36 hours prior to imaging, and then photobleaching using a 60x oil immersion objective (CFI Apo TIRF, NA = 1.49). Laser lines (488 nm) were adjusted to an output power of 8 mW. The instrument (A1, Nikon) was set to a zoom of 60X, a region of interest of 23.94 µm 2 (5.7 µm x 4.2 µm), a photomultiplier voltage of 80 V with a scan time of 1.5 seconds per frame. Nuclei having approximately the same dimensions and intensity under the fixed instrument settings were chosen for photobleaching assays. T1/2 bleach is time to bleach to 50% emission intensity under laser. T1/2 bleach of GFPSpark® under 488 nm State Sapphire Solid laser is 12.4 s and EGFP is 9.2s.The photostability of GFPSpark® is better than EGFP.