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Glutathione (GSH) Probes

Ursa BioScience offers a range of patent protected water soluble and highly photostable probes (dye molecules) for both the rapid detection and quantitation of Glutathione (GSH). Our probes can detect GSH in an absorption (colorimetric); absorption ratiometric (quantitation); fluorescence; or fluorescence ratiometric (quantitation) manner, without the need for enzymes and long wait times.

Our probes (dye molecules) are very bright, easy to use and provide highly reproducible results.

We offer our probes in standard vials, which can be simply reconstituted by adding solvent / buffer, or alternatively in a 96-well kit format.

  • Absorption - A colorimetric GSH detection method.
  • Absorption Ratiometric – A colorimetric and quantitative GSH detection method.
  • Fluorescence – Fluorescence based detection of GSH.
  • Fluorescence Ratiometric – A quantitative fluorescence method for GSH.

GSH Red™

Glutathione Red (GSH Red™) is a unique water soluble, highly fluorescent probe that can both detect and quantitate Glutathione (GSH). Its long absorption and emission wavelengths make it ideal for flow cytometry, microscopy and HCS imaging. GSH Red is ideal for measuring the cellular levels of reduced glutathione and can readily be used with cationic surfactants such as CTAB and BC.

Absorption spectra and ratiometric plot recorded for GSH Red

Figure 1. (A) Absorption spectra recorded for 2.5 µM GSH Red™ with 5 µM Glutathione. As a function of time the absorption band at 580 nm dramatically increases, giving GSH Red™ its distinct red color in the presence of glutathione. (B) Absorption wavelength ratiometric plot, constructed by taking the absorption at 580 nm divided by that at 360 nm. The plots also show the specificity towards glutathione as compared to other biological thiols.

 

Figure 1 shows the absorption spectrum of GSH Red™ (2.5 µM) in the presence of 5 µM Glutathione dissolved in phosphate buffered saline solution at pH 7.4. As a function of time (< 10 mins), GSH Red™ turns the solution an intense red color with an absorption maximum ~590 nm. GSH Red™ is specific to Glutathione, Figure 1 Right, showing a significant response as compared to L-Cysteine (L-Cys) and Homocysteine (HCys). A short video shows the specificity and rapidity of GSH Red™ to Glutathione. The absorption ratiometric plots are simply generated by dividing the absorption values at 580 nm, divided by the absorption values at 360 nm as a function of time. GSH Red™ can be used in both an absorption mode (colorimetric), or in an absorption ratiometric manner. For quantitation, absorption values at 10 mins after mixing are typically used.

Visual comparison

Figure 2. Photograph showing the specificity of GSH Red™ to glutathione Vs other biological thiols, taken after (A) 2 minutes and (B) 10 minutes. The concentration of GSH Red™ was 5 µM. The concentration of L-Cys, HCys and Glutathione was 10 µM. All cuvettes contained 2 mM of CTAB. The control sample contains no thiols, only PBS/CTAB and GSH Red™.

 

Glutathione specifically reacts with GSH Red™ very quickly (much faster than Enzyme based methods) allowing for the rapid both detection and quantitation of Glutathione. Figure 3A shows the rapid response of GSH Red™ to Glutathione Vs time.  By taking the ratio of the absorbance at 580 / 360 nm, a ratiometric plot readily allows for Gluathione concentration to be accurately quantitated, Figure 3B. This approach can be applied to virtually and cell or lysate type, in either a cuvette or 96-well plate format.

Absorption spectra and ratiometric plot recorded for GSH Red

Figure 3. (A) Time-dependent ratiometric plots (from absorption spectra) constructed for differerent concentrations of glutathione, i.e by dividing the peak value at 580 nm with that at 360 nm. The concentration of GSH Redtm was 2.5 uM with 2 mM CTAB. (B) Ratiometric value observed at 10 minutes plotted against the concentration of glutathione. Subsequently, the A580/A360 value allows glutathione to be quantitatively determined.

 

GSH Red™ shows brilliant red fluorescence when excited around 500 nm (Excitation range 400-600 nm), with fluorescence from 570-680 nm.

Figure 4. (A) Fluorescence emission of 2.5µM GSH Red™, excited at 520 nm in the presence of 5 µM Glutathione and 2 mM CTAB (Cetyltrimethyl Ammonium Bromide). (B) The corresponding change in fluorescence as a function of time. The plots also show the specificity towards glutathione as compared to other biological thiols, also used at 5 µM concentration.

 

Similar to the absorption response, the fluorescence increases with time. Values at time = 10 mins after mixing are typically quoted for analytical measurements.

We recommend our 500DLP dichroic long pass filter, which can readily be mounted in a microscope filter cube for use with GSH-Red™.

GSH Red™ in a 96-well Format

We offer GSH Red™ in a convenient 96-well kit format. GSH Red™, 2 bottles of CTAB surfactant and a clear bottom black 96-well plate are supplied, allowing for GSH detection in any 3 formats: Absorption (Colorimetric); Absorption Ratiometric (Quantitative) and Fluorescence based detection.  Enough reagents are supplied for 98 – 300 µL assays, or 294 – 100 µL assays.

Go to the online store for the price of this kit.

GSH Red™ in Pre-Loaded Cuvettes

We offer GSH Red™ (and CTAB) already conveniently loaded into a box of 100 plastic cuvettes (transmission down to 285 nm). These cuvettes allow for GSH detection in any 3 formats: Absorption (Colorimetric); Absorption Ratiometric (Quantitative) and Fluorescence based detection. Simply add 3 ml (3cm3) of your sample to each cuvette and measure, it’s that simple.

Go to the online store for a price for this kit.

GSH Red FR™

Our GSH Red Fluorescence Ratiometric (GSH Red FR™) vials, contain a mixture of both GSH-Red™ and a second fluorophore (an internal reference standard), both fluorophores can readily be excited from 520-560 nm. The second internal standard fluorophore is unperturbed by the presence of biological thiols, allowing for quantitative fluorescence ratiometric measurements to be undertaken by dividing the intensity of the GHS Red™ peak by that of the internal reference standard. This ratiometric approach is particularly useful for quantitative GSH measurements. GSH Red FR™ is ideal for measuring the cellular levels of reduced glutathione and can readily be used with cationic surfactants such as CTAB and BC.

GSH Red FR™ is sold dry and is simply reconstituted by adding solvent. Specific details supplied in the data sheets.

Figure 5. (A) Time-dependent emission spectra recorded for a 2.5 μM GSH Red solution (PBS mixed 2.5 mM CTAB) after glutathione (5 μM) was added. The excitation wavelength was 560 nm. Note that the emission spectra from the internal reference, centered at 710 nm, is shown on an expanded scale. (B) Ratiometric emission plot constructed by dividing peak intensities for GHS Red and the internal reference.

 

Advantages of the GSH Red FR™ Fluorescence Ratiometric Approach

It is widely recognized that ratiometric or lifetime-based methods offer intrinsic advantages for both chemical and biomedical sensing [1,2]. Fluorescence intensity measurements alone are typically unreliable away from the laboratory and can require frequent calibration/s due to a variety of chemical, optical or other instrumental related factors [1,2]. Unfortunately, while fluorescent probes are known to be very useful for many applications [1,2], such as in fluorescence microscopy, DNA technology and fluorescence sensing, most sensing fluorophores only display changes in intensity in response to analytes, and relatively few wavelength ratiometric and lifetime based probes are available [1,2]. GSH Red FR™ allows quantitative glutathione measurements to be undertaken, without the worry of instrumental artifacts, such as intensity drift.

Cited References

[1] Valeur, B and Berberan-Santos, MN, Eds, "Molecular Fluorescence", second edition, Wiley-VCH, Weinheim, Germany 2012.

[2] Z. Gryczynski, I. Gryczynski, J. R. Lakowicz, Methods in Enzymology, (2002), 360, 44.

 

 

 

 

 

 

 

The probe GSH Red shines up in the prescence of Glutathione

GSH Red™ is an exceptionally sensitive probe for glutathione. The probe emits an intense red fluorescence that can readily be detected by any standard fluorometer or microscope.

 

Video demonstrating both the visual color change and specificity of GSH Red™ for Glutathione as compared to other biological thiols.

 

contour

3D Ex/Em contour emission spectra recorded for GSH Red™ in pbs buffer with 2 mM CTAB.

 GSH Red™ is supplied dry in a prepacked vial for easy reconstitution. A 2 mM CTAB solution is also supplied with each vial order.

GSH Red™ is supplied dry in a prepacked vial for easy reconstitution. A 2 mM CTAB solution is also supplied with each vial order. Visit our Online Store for current pricing.

 

GSH Red™ 96-well kit contains i) GSH Red™, ii) Clear-bottom, Black, 96-well plate and iii) Two bottles of CTAB.

GSH Red™ 96-well kit contains i) GSH Red™, ii) Clear-bottom, Black, 96-well plate and iii) Two bottles of CTAB. Check out the Online Store for pricing.

red

GSH Red 100 Cuvette Kit contains 100 preloaded cuvettes in a convenient box set. Both GSH Red and CTAB are preloaded, so all that is needed is 3 ml of your sample, then measure. Check out the online store for pricing.