GSH/GSSG Ratio Detection Assay Kit (Fluorometric - Green) (ab138881)

Overview

  • Product name
    GSH/GSSG Ratio Detection Assay Kit (Fluorometric - Green)
    See all Glutathione kits
  • Detection method
    Fluorescent
  • Sample type
    Urine, Serum, Plasma, Tissue Extracts, Cell Lysate
  • Assay type
    Quantitative
  • Sensitivity
    10 nM
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mammal
  • Product overview

    Abcam's GSH/GSSG Ratio Detection Assay Kit (Fluorometric - Green) (ab138881) provides an ultrasensitive assay to quantitate glutathione in the sample.


    There are quite a few reagents or assay kits available for quantitating thiols in biological systems. However, most commercial kits either lack sensitivity or have tedious protocols. ab138881 uses a proprietary non-fluorescent dye that becomes strongly fluorescent upon reacting with GSH. With a one-step fluorimetric method, the kit can detect as little as 1 picomole of GSH or GSSG in a 100 µL assay volume. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and readily adapted to automation without a separation step. Its signal can be easily read by a fluorescence microplate reader at Ex/Em = 490/520 nm.


    NOTE: For measuring GSH Standard only, there is enough reagent provided to perform 200 tests.


    This product contains a DMSO-soluble probe. If you prefer to use a water-soluble probe, we recommend using GSH/GSSG Ratio Detection Assay Kit II (Fluorometric - Green) (ab205811).

  • Notes

    Glutathione (GSH) is a tripeptide that contains L-cysteine, L-glutamic acid and glycine. It is the smallest intracellular protein thiol molecule in the cells, which prevents cell damage caused by reactive oxygen species such as free radicals and peroxides. Glutathione exists in reduced (GSH) and oxidized (GSSG) states. Reduced glutathione (GSH) is a major tissue antioxidant that provides reducing equivalents for the glutathione peroxidase (GPx) catalyzed reduction of lipid hydroperoxides to their corresponding alcohols and hydrogen peroxide to water. In the GPx catalyzed reaction, the formation of a disulfide bond between two GSH molecules generates oxidized glutathione (GSSG). The enzyme glutathione reductase (GR) recycles GSSG to GSH with the simultaneous oxidation of β-nicotinamide adenine dinucleotide phosphate (β-NADPH2). In healthy cells, more than 90% of the total glutathione pool is in the reduced form (GSH). When cells are exposed to increased levels of oxidative stress, GSSG accumulates and the ratio of GSSG to GSH increases. An increased ratio of GSSG-to-GSH is an indication of oxidative stress. The monitoring of reduced and oxidized GSH in biological samples is essential for evaluating the redox and detoxification status of the cells and tissues against oxidative and free radicals mediated cell injury.

  • Tested applications
    Suitable for: Functional Studiesmore details

Properties

Associated products

Applications

Our Abpromise guarantee covers the use of ab138881 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Functional Studies Use at an assay dependent concentration.

Images

  • GSH in reduced state measured in cell lysates showing quantity (umol) per 1 mln cells.

    Samples with the concentration of 1e7-1e8 cells/mL were used. Samples were diluted 10-1000 fold.

  • Total GSH measured in cell lysates showing quantity (umol) per 1 mln cells.

    Samples with the concentration of 1e7-1e8 cells/mL were used. Samples were diluted 10-1000 fold.

  • GSH in reduced state measured in tissue lysates showing quantity (umol) per miligram of extracted protein of tested sample.

    Protein concentration for samples varied from 6 mg/mL to 16 mg/mL. Samples were diluted 10-100 fold.

  • Total GSH measured in tissue lysates showing quantity (umol) per miligram of extracted protein of tested sample.

    Protein concentration for samples varied from 6 mg/mL to 16 mg/mL. Samples were diluted 10-100 fold.

  • GSH in reduced state measured in biological fluids showing concentration (uM) in tested samples. Human samples were diluted 10 fold. Rat sample was diluted 10-1000 fold.

  • Total GSH measured in biological fluids showing concentration (uM) in tested samples. Samples were diluted 10-100 fold. 

  • Total GSH dose responses were measured with ab138881 in a black 96-well plate using a fluorescence microplate reader. 50 µl of GSSG standards (0.01 to 5 µM), GSH-containing samples or blank control were added into each well, and then 50 µl of Total GSH Reaction Mixture was added. Fluorescence intensity was measured at Ex/Em = 490/520 nm after 30 minutes incubation.
  • Reduced GSH dose responses were measured in a black 96-well plate with ab138881 using a fluorescence microplate reader. 50 µl of GSH standards (0.01 to 5 µM) or blank control was added into each well, and then 50 µl of GSH Assay Mixture was added. The fluorescence intensity was measured at Ex/Em = 490/520 nm after 30 minutes incubation.

Protocols

References

This product has been referenced in:
  • Persson T  et al. Apolipoprotein E4 Elicits Lysosomal Cathepsin D Release, Decreased Thioredoxin-1 Levels, and Apoptosis. J Alzheimers Dis 56:601-617 (2017). Read more (PubMed: 28035917) »
  • Tsai WT  et al. Mycotoxin Patulin Suppresses Innate Immune Responses by Mitochondrial Dysfunction and p62/Sequestosome-1-dependent Mitophagy. J Biol Chem 291:19299-311 (2016). Read more (PubMed: 27458013) »

See all 7 Publications for this product

Customer reviews and Q&As

The lab has sent an alternative protocol that might be useful for you:

1) For GAM: Alternatively, one can make GSH Assay Mixture by adding 100X Thiol Green stock solution with Assay Buffer proportionally.

2) For TGAM: Alternatively, o...

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We measured GSH levels in OVCAR4 cells tranfected with scrambled siRNA or CSE siRNA. We used Cell Lytic M lysis buffer for lysis followed by deproteinisation. The assay was straightforward and the standards gave perfect plots for quantifying amounts of GSH in our samples. We used I million cells for each sample.
Scope of improvement: Providing an assay compatible lysis buffer in the kit will be appreciable.
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Verified customer

Submitted Dec 12 2017

Le protocole indique de neutraliser les échantillons en ajoutant du NaHCO3 goutte à goutte jusqu'à ce que le pH soit de 4-6. La concentration de NaHCO3 n'est donc pas très importante. Si vous utilisez une solution concentrée, vous ajouterez moins de go...

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Detection of GSH levels in equine fibroblasts

Average Average 3/5 (Ease of Use)
Abreviews
I wanted to measure GSH levels in equine fibroblasts and decided to try the abcam kit. Upon arrival the kit required aliquoting and reconstituting but once this was done it was easy to set up the assay. Some reagents aren't included in the kit (cell lysis buffer and deproteinisation kit) but these are easily ordered from abcam as per the protocol.
This assay worked on equine cells and gave similar values as per other cell types provided in the handbook. The kit did however give similar readings for different sample dilutions (before calculations) and therefore raised some concern regarding sample preparation (I used the mammalian cell lysis buffer and deproteinisation kit from abcam).
The calculations are quite straight forward but not explained particularly well in the handbook, this would be clarified using a working example perhaps.
Figure legend:
Total GSH and reduced GSH levels (uM per 1million cells) in equine fibroblast lysates. Measurements taken at 1/100 dilution of samples. Mean +/- SEM, samples were run in duplicate. Samples were cultured in different glucose concentrations (- :5.5mM, + :25mM).
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Submitted Jun 09 2016



For serum sample, you need to deproteinize the sample first before analysis. Here is the brief instruction:



1. Freeze serum samples immediately upon acquisition and keep frozen until ready for processing.

2. Take 1...

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GSH-GSSG kit

Good Excellent 5/5 (Ease of Use)
Abreviews
The FAQ section is the only place to find info about sample prep. I used the Metaphosphoric acid method, and I sonicated the samples after resuspending the cells in this acid. (I used a cup horn sonicator, direct contact, full power, for 10 seconds x 3, so that was very much high power). The assay worked well. I don't like the fact that the GSSG probe comes in a big bottle and that when you make the 25x stock solution in this bottle you have to work very hard to get the 200uL of liquid to come in contact with all the little bits of powder. I ended up solving this problem by putting the bottle in a TC centrifuge for a couple of spins and eventually got all the bits of powder down to the bottom of the bottle where they could be dissolved in the 200uL of liquid. Otherwise I loved the ease of use of the kit. MUCH easier than a GSH-OPA assay! The instruction manual has places where it should be re-written to make more sense and should include info about sample prep.
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Verified customer

Submitted Dec 17 2014

Our in house experiments showed increase in background with Triton X-100 which is why we recommend auto-fluorescent free Triton-X 100 or simply avoiding this lysis buffer.
We recommend following sample preparation protocol:
GSH is labile and ...

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You can use 0.5% NP40 made up in PBS pH6.0 to lyse your cells. Please do not use the assay buffer to lyse your cells as it contains no detergent.

In general, we do not deproteinate our samples however, you may include this step if you wish. Read More



The concentrations of Total GSH standard solutions should be twice the concentrations of GSSG standard solutions as 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1563, and 0 μM”.

This is because after enzyme reaction, GSSG will be con...

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Regarding the GSSG probe could be used with the thiol assay ab112158, the laboratory that developed these assays replied that, yes, the GSSG probe is a reducing agent that reduces cysteine double bonds. So it can be used to detect the redox ratio of ti...

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