Green Fluorescent Protein GFPSpark?

The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to blue light. GFPSpark? is an improved variant of GFP. It possesses bright green fluorescence (excitation/ emission max = 487 / 508 nm) that is visible earlier than the fluorescence of other green fluorescent proteins. GFPSpark? consists of 245 amino acids, and with a calculated molecular weight of 26.8 kDa. GFPSpark? is mainly intended for applications where fast appearance of bright fluorescence is crucial. Its amazing ability to generate a highly visible, efficiently emitting internal fluorophore is both intrinsically fascinating and tremendously valuable. It is specially recommended for cell and organelle labeling and tracking the transcriptional activities of promoters.

Advantages of Green Fluorescent Protein GFPSpark ?

Bright green fluorescence High pH-stability Fast maturation Intellectual property
Recommended for gene expression analysis, cell and organelle labeling
Proven suitability to generate stably transfected cell lines
  • Fluorescence spectra of GFPSpark?
  • SDS PAGE analysis
  • GFPSpark? ? product image

GFPSpark? Protein:Brighter Green Fluorescence

GFP Improved EGFP Optimized GFPSpark?
Bright Brighter Brightest

GFPSpark? Main Characteristics

  Fluorescence color Extinction coefficient (M-1cm-1) Quantum yield Brightness Pka Structure Maturation rate at 30℃ t1/2 bleach(s)
GFPSpark? Green 47000 0.62 29.14 4.5 Monomer Superfast 12.4
EGFP Green 39000 0.6 23.4 5 Monomer Fast 9.2

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 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.

pH Sensitivity

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.

Photobleaching Assays

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.

GFPSpark? Gene Expression

GFPSpark?, EGFP, commercial GFP1 or commercial GFP2 recombinant plasmids were constructed on pCMV3 vector. Transiently transfected 293 H cells, the green fluorescent signals can be detected in cytoplasm under the excitation channel of 455~495 nm by Microscopy. GFPSpark? gives the brightest signal 24 hrs, 48 hrs and 96 hrs after transfection.

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