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
  • Product overview

    GSH/GSSG Ratio Detection Assay Kit (Fluorometric - Green) (ab138881) provides an ultrasensitive assay to quantitate glutathione in mammalian samples.


    This kit uses a proprietary non-fluorescent dye that becomes strongly fluorescent upon reacting with GSH. With a one-step fluorimetric method, the assay 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.

Properties

Associated products

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:
  • Serbulea V  et al. Macrophages sensing oxidized DAMPs reprogram their metabolism to support redox homeostasis and inflammation through a TLR2-Syk-ceramide dependent mechanism. Mol Metab 7:23-34 (2018). Read more (PubMed: 29153923) »
  • Szelechowski M  et al. Metabolic Reprogramming in Amyotrophic Lateral Sclerosis. Sci Rep 8:3953 (2018). Read more (PubMed: 29500423) »

See all 14 Publications for this product

Customer reviews and Q&As

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...

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For lysis, 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.

The deproteinization step is optional, however, we do find that it reduces interferenc...

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We have published ab205811 as an improved version of ab138881. The new product has a water-soluble probe instead of DMSO-soluble, so it should easier to solubilize. For the time being, ab138881 will remain in the catalogue as it gives slightly less sam...

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No direct experiences on total blood but should be OK theoretically. The following may help:

1. add equal vol of ice-cold 10% Metaphosphoric acid (MPA) working solution. mix well, let sit at RT for 5 min.
2. Centrifuge at >2,000xg at 4°C ...

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We recommend to use 0.5% NP40 as cell lysis buffer. The Assay buffer does not contain any detergent and therefore it is not suitable to be used for lysing the cells.

The GSH is very easy to be oxidized. If GSH standard signal is lower than GSSG (or GAM is higher than total GAM), the GSH may be oxidized.

DTT or 2-mercaptoethanol cannot be used for this case. In our kit, GSSG probe is an enzyme-based mixture with other additives that can convert GSSG to GSH, which can be recognized by the probe and generate green fluorescence after reaction. DTT or 2-me...

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Abreviews
I used GSH/GSSG ratio detection assay kit to measure GSH levels in the BV-2 cells treated with LPS and astaxanthin. Standard curve was perfect to examine total GSH levels and GSH/GSSG ratio. We used 1 million cells for each samples that were appropriate to detect GSH levels. The handbook was great to perfome this assay and calculate our results.
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Ms. Ji Hye Han

Verified customer

Submitted Apr 06 2018

Detection of GSH/GSSG ratio in plants

Good Good 4/5 (Ease of Use)
Abreviews
I used this kit to measure the GSH/GSSG ratio in plants after treatment with high light. For this, 6-week- old Arabidopsis thaliana (Col) plants were treated with high light (1000 µmol m-2 sec-1) for 0, 2 and 15 minutes and immediately frozen in liquid nitrogen. Three plants were used for each time point. Each sample was then grinded in liquid nitrogen and 20mg of frozen powder was taken for the assay. I also purchased the mammalian lysis buffer from abcam. 400µl of the lysis buffer was immediately added to the sample after weighing. The rest of the assay and calculations were done according to the protocol provided.
Username

Feroza Kaneez

Verified customer

Submitted Jan 24 2018

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.
Username

Abcam user community

Verified customer

Submitted Dec 12 2017

1-10 of 16 Abreviews or Q&A

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