MetalloFluor™ Series
[Copper ion detecting probe]
495-540 nm：Green
Copper is one of the trace metals essential for living organisms. Copper is an essential cofactor for enzyme activities of cytochrome c, superoxide dismutase, and tyrosinase. CopperGREEN is a fluorescent probe to detect Cu+, which is a dominant redox state of copper in an intracellular reducing environment.
Available through Merck KGaA (Darmstadt, Germany) as:
SCT041 BioTracker™ Green Copper Dye
| Code No. | Product Name | Size | Merck CAT No. |
Merck ( Millipore / Sigma Aldrich ) Product Name |
|---|---|---|---|---|
| GC902 | CopperGREEN™ | 50 nmol × 5 | SCT041 | BioTracker Green Copper Dye |
CopperGREEN™
Product Information
CopperGREEN is a colorless material with almost no fluorescence. When CopperGREEN chelates Cu+ ion, it is easily degradated to generate fluorescent material with excitation maximum, ~480 nm and emission maximum, 510 nm. Fluorescence remains after mild fixation because its reaction is irreversible. It does not react with other trace metals in cells including Cu2+, Mn2+, Co2+, Ni2+, Fe+, Fe2+, Zn2+.
| Product name |
target | reaction | Absmax (nm) | FLmax (nm) |
| CopperGREEN | Cu+ | irreversible | ~480 | 510 |
Upon the reaction with Cu+, its fluorescence intensity at 510 nm increases >100 times to that before the reaction (left).
Reaction properties indicate that CopperGREEN reacts with Cu+ ions at molar ratio of 1:1. Reaction takes ~90 minutes to saturate. (right)
- (left) Fluorescence spectra of 5 µM CopperGREEN before and after the reaction with Cu+. CopperGREEN was reacted with 100 μM [CuI(CH3CN)4]PF6 at 37℃ for 2 hours in a buffer solution of 50 mM HEPES (pH 7.2), 2 mM glutathione, and 0.5% DMSO as a cosolvent.
- (right, top) Fluorescence intensity of 1 µM CopperGREEN depending on the concentration of Cu+, which was measured using a microplate reader with excitation wavelength of 470 nm, emission wavelength of 510 nm, excitation bandwidth of 9 nm, emission bandwidth of 20 nm. CopperGREEN was reacted with [CuI(CH3CN)4]PF6 at 37℃ for 2 hours in a buffer solution of 50 mM HEPES (pH 7.2), 2 mM glutathione, and 0.1% DMSO as a cosolvent.
- (right, bottom) Time course of 1 µM CopperGREEN reaction with 100 μM Cu+. Fluorescence was measured by using fluorescence spectrophotometer with excitation wavelength of 470 nm, emission wavelength of 510 nm.
Fluorescence increase is observed in the presence of Cu+ ion, but not in the presence of other trace metals that exists in biological environments. Fluorescence increase is not also observed in the presence of reactive oxigen species.
- Fluorescence of 1 µM CopperGREEN in 0.05 M HEPES buffer (pH7.2) were measured after incubation of with the following reagents at 37℃, 120 min. Either metal ions of 20 μM supplemented with 2 mM glutathione, or ROS generating reagents as described later was used. In Cu2+ condition, glutathione was not used.
- Fluorescent intensities were measured at 510 nm, with excitation at 470 nm by using a microplate reader.
ROS generating conditions
- H2O2: 300 µM H2O2
- OCl－: 300 µM NaOCl
- ・OH: 20 µM Fe(ClO4)2, 200 µM H2O2
- none: 0.05 M HEPES buffer (pH 7.2) as a control.
CopperGREEN™
An example live-cell Imaging of copper
Fluorescence image of CopperGREEN (green) was overlaid to DIC image.
HeLa cells cultured in a culture medium (DMEM + 8% FBS, penicillin and streptomycin) supplemented with 200 µM CuCl2 were rinsed with PBS containing 200 µL EDTA for 2 times to remove extracellular copper ions. Then cells were reacted with 5 μM CopperGREEN at 37℃ 5% CO2 for 3 hours. For the fluorescence observation, the medium was exchanged with HBSS to reduce the background.
※ To reduce nonspecific staining, add 10 mM NH4Cl or 100 nM bafilomycin A1 to the medium 30 minutes before the staining and during the staining. This treatment inhibit acidification of lysosomes and reduces nonspecific oxidization of CopperGREEN in acidic environments. (10 mM NH4Cl was used in the above example.)
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