Orange/Red Fluorescencent Protein OFPSpark®

OFPSpark® is a red (orange) fluorescent protein (excitation/emission maxima are 549 and 566 nm, respectively) derived from DsRed. OFPSpark® is consist of 231 amino acids and the calculated molecular weight of OFPSpark® is 26.4 kDa. Possessing high photostability and pH stability.

Advantages of Orange/Red Fluorescencent Protein OFPSpark®

Bright red (orange) fluorescence High pH-stability
Superior photostability stability Fast maturation
Intellectual property Recommended for gene expression analysis, cell and organelle labeling
  • Fluorescence spectra of OFPSpark®
  • SDS PAGE analysis
  • OFPSpark® product image

OFPSpark® Protein:Bright Red (Orange) Fluorescence

DsRed Commercial Orange Red OFPSpark®
Tetramer
not suitable for fusion protein expression
Monomer Brighter

OFPSpark® Main Characteristics

  Fluorescence color Extinction coefficient
(M-1cm-1)
Quantum yield Brightness Pka Structure Maturation rate at 30℃ t1/2 bleach(s)
OFPSpark® Red(Orange) 84000 0.69 58 6.2 Monomer Fast 4.7
Commercial orange Red Red(Orange) 49000 0.6 29.4 6.8 Monomer Fast 4.8

Fluorescence Spectra Characteristics

A)Extinction Coefficient:

B)Brightness

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.

Maturation Kinetics

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 .

pH Sensitivity

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.

Photobleaching Assays

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.

OFPSpark® Gene Expression

OFPSpark®, commercial RFP or commercial orange RFP recombinant plasmids were constructed on pCMV3 vector.The plasmids transiently transfected into 293 H cells. The fluorescent signals detect by fluorescent microscope.The orange fluorescent signals detect under the excitation channel of 503.5~547.5 nm, and the red fluorescent signals detecte under the excitation channel of 532.5~587.5 nm. OFPSpark® gives the brightest red signal 24 hrs, 48 hrs and 96 hrs after transfection.