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 OFPSpark® is 549nm，and the emission spectra is 566nm. The quantum yield of OFPSpark® is 0.69 and commercial orange Red is 0.60.The extinction coefficient of OFPSpark® is 84000 and commercial orange Red is 49000. The extinction coefficient of OFPSpark® is better than commercial orange Red.
To compare maturation of OFPSpark® and commercial orange Red , HeLa cells were transiently transfected with OFPSpark® or commercial orange Red under the control of CMV promoter. The DNA concentrations were equalized before transfection. Cells were photographed using fluorescent microscope after different periods of cultivation. The maturation of OFPSpark® is same to commercial orange Red .
The pH sensitivity of OFPSpark® was determined in a 96-well format by adding 100 µl of dilute OFPSpark® in a weakly buffered solution to 100 µl of strongly buffered pH solutions in triplicate (total 200 µl per well) for pHs from 2 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 OFPSpark® is 6.2 and commercial orange Red is 6.8. The pH stability of OFPSpark® is better than commercial orange Red.
Laser scanning confocal microscopy (LSCM) photobleaching experiments were conducted with the OFPSpark®. 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 (561 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 OFPSpark® under 561 nm State Sapphire Solid laser is 4.7s and commercial orange Red is 4.8s.The photostability of OFPSpark® is same to commercial orange Red.