Quantification of Reduced (GSH) and Oxidized (GSSG) Glutathione

∼ Feature ∼

  1. Optimized Enzymatic recycling system
  2. High reproducebility for detecting GSH, GSSG and total glutathione individually
  3. Contain optimzed Masking Reagent for exclusive measurement of GSSG
  4. Easy and Simple procedure; takes 1-2 hours


 

MSDS


Product Description
Glutathione (γ-L-glutamyl-L-cysteinylglycine) is a tripeptide present in the body, and it is involved in antioxidation, drug metabolism, and other as enzyme substrate of glutathione peroxidase, glutathione S-transferase, and thiol transferase, etc. Glutathione is usually present as reduced form (GSH), but GSH is converted into its oxidized form (GSSG) by stimulation such as oxidative stress. Therefore, the ratio of GSH and GSSG has been noted as index of oxidative stress.
The GSSG/GSH Quantification kit contains Masking Reagent of GSH. The GSH can be deactivated in the sample by simply adding the Masking Reagent. Therefore, temp-only the GSSG is detected by measuring the absorption (λmax = 412 nm) of DTNB (5,5 Edithiobis (2-nitrobenzoic acid) using the enzymatic recycling system. Also, GSH can be determined the quantity by subtracting GSSG from the total amount of glutathione.
The kit is limited to quantifying GSH/GSSG concentration from 0.5 μmol/l to 50 μmol/l and GSSG concentration from 0.5 μmol/l to 25 μmol/l.


Principle


Selective Quantification
Although conventional masking reagent, 2-Vinylpyridine(2-VP), interferes the reaction of GSSG measurement, Dojindo’s masking reagent does not interfere the reaction of GSSG measurement. Therefore, the exact ratio of GSSG and GSH is obtained with Dojindo GSSG/GSH detection Kit.

Measurement of GSSG with or without GSH masking reagent


Interference
Reducing agents such as ascorbic acid, β-mercaptoethanol, dithiothreitol (DTT) and cysteine, or thiol reactive compounds such as maleimide compounds, interfere with the glutathione assay. Therefore, SH compounds, reducing agents and SH reactive materials should be avoided during the sample preparation.


Required Equipment and Materials
- Plate reader (405 or 415 nm filter)
- 96-well microplate
- Incubator (37ºC)
- 20 µl and 200 µl pipettes, a multi channel pipette
- 5-Sulfosalicylic Acid (SSA) Solution
- Ethanol


Assay Procedure



Contents of the Kit
G257-10 (200 tests)

Enzyme solution

: 50 μl, 1 vial

Coenzyme : 2 vials

Buffer solution

: 60 ml, 1 bottle

Substrate (DTNB) : 4 vials

Standard GSH

: 1vial

Standard GSSG : 1vial

Masking reagent

: 20 μl, 1 vial


Storage Condition : 0-5oC, protect from light
Shipping Condition : ambient temperature

Literature used "GSSG/GSH Quantification Kit"

Diurnal Variation of cadmium-induced mortality in mice
N. Miura, Y. Yanagiba, K. Ohtani, M. Mita, M. Togawa, and T. Hasegawa, J. Toxicol. Sci., 2012, 37(1), 191
<Detailed information on this article>
- Measuring object: Hepatic GSH
- Sample: Liver (Mice)
- Preparation of sample
1) Homogenize liver sample in 5% SSA for 30 seconds in ice-water bath.
2) Centrifuge the homogenates consisting of 100 mg liver in 1 ml (10%) at 8,000 x g for 10 min. at 4°C to remove proteins.
3) Assay with supernatants for GSH using GSSG/GSH Quantification Kit according to the manufacture's instruction.


<Detailed information on this article>
- Measuring object: GSH
- Sample: Parotid acinar cells
- Preparation of sample
1) Collect parotid acinar cells by centrifugation at 10,000 g for 15 s and immediately mixed with 160 ul of 10 mM HCl.
2) Freeze and thraw the mixture three times over, then mix with 40 ul of 5% SSA and then centrifuge at 8,000 g for 10 minutes.
3) Collect the supernatant and dilute twice.

Chloroplast DNA Replication Is Regulated by the Redox State Independently of Chloroplast Division in Chlamydomonas reinhardtii Y. Kabeya, and S. Miyagishima, Plant Physiol., 2013, 161, 2102

<Detailed information on this article>
- Measuring object: GSH and GSSG
- Sample: Chlamydomonas reinhardtii
- Preparation of sample
1) Collect Chlamydomonas reinhardtii cells by 1,000g for 5 min and wash with PBS.
2) Resuspended in 5% SSA solution and disrupted by sonication.
3) Collect the supernatants by 15,000g for 5 min.
4) Isolate chloroplst by 700g for 5 min and wash with 50 mM HEPES-KOH, pH 7.5, containing 300mM sorbitol.
5) Resuspend in 5% SSA solution and centrifuge at 15,000g for 5 min

Selected publications
ER Stress Cooperates with Hypernutrition toTrigger TNF-Depe. Cancer Cell. 2014;26:331-343.
Purpose: Determine indicator of ER Stress in Liver


Mechanisms of cadmium-inducedchronotoxicity in mice. The journal of toxicological. 2013;38:947-957.
Purpose: Determine the GSH/GSSG ratio in cadmium exposed Liver


Hepatitis C Virus Core Protein SuppressesMitophagy by Interacting with Parkin in the Context of MitochondrialDepolarization. The American Journal of Pathology. 2014;184:3026-3039.
Purpose: Measure mitochondrial oxidative status in liver

Possible involvement of glutathione balance disruption in dihydropyrazine-induced cytotoxicity on human hepatoma HepG2 cells.The journal of toxicological.2012;37:1065-1069.
Purpose: Investigate fluctuation of GSH/GSSG ratio in the cytotoxicity HepG2 Cells

Induction of heme oxygenase-1 contributes to survival of Mycobacterium abscessus in humanmacrophages-likeTHP-1cells.Redox Biology. 2015;4:328-339.
Purpose: Determine redox state in macrophages


Age-related changes in salivary biomarkers.Journal of Dental Sciences. 2014;9:85-90.
Purpose: Measure GSH/GSSG ratio in salivary

Protective effects of hydrogen sulfideanions against acetaminophen-induced hepatotoxicity in mice. The journal oftoxicological. 2015;40:837-841.
Purpose: Measure glutathione of Hepatocyte


Other References

1. N. Kubota, et al., A high-fat diet and multiple administration of carbon tetrachloride induces liver injury and pathological features associated with non-alcoholic steatohepatitis in mice. Clin Exp Pharmacol Physiol. 2013; 40(7): 422-30
2. T. Tomofuji, et al., Supplementation of broccoli or Bifidobacterium longum-fermented broccoli suppresses serum lipid peroxidation and osteoclast differentiation on alveolar bone surface in rats fed a high-cholesterol diet. Nutr Res. 2012; 32(4): 301-7
3. T. Miyayama., et al., Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate. Toxicology. 2013; 8;305: 20-9.
4. H. Miwa, et al., Leukemia cells demonstrate a differen t metabolic perturbation provoked by 2-deoxyglucose. Oncol Rep. 2013; 29(5): 2053-7.
5. K. Unno, et al., Acute enhancement of non-rapid eye movement sleep in rats after drinking water contaminated with cadmium chloride. J Appl Toxicol. 2013 24.
6. Y. Ishihara, et al., Tributyltin induces oxidative stress and neuronal injury by inhibiting glutathione S-transferase in rat organotypic hippocampal slice cultures. Neurochem Int. 2012; 60(8): 782-90.
7. H. Miyamoto, et al., Thermophile-fermented compost as a possible scavenging feed additive to prevent peroxidation. J Biosci Bioeng. 2013; 116(2): 203-8.
8. E. Taniai, et al., Ochratoxin A induces karyomegaly and cell cycle aberrations in renal tubular cells without relation to induction of oxidative stress responses in rats. Toxicology Letters, October 2013.
9. G. Tian, et al., Ubiquinol-10 Supplementation Activates Mitochondria Functions to Decelerate Senescence in Senescence Accelerated Mice. Antioxidants & Redox Signaling, October 2013.


Example for Sample preparation

Tissue (100 mg)
1) Homogenize the tissue in 0.5-1.0 ml of 5% SSA.
2) Centrifuge the homogenized tissue sample at 8,000 xg for 10 minutes.
3) Transfer the supernatant to a new tube and add double-deionized H2O (ddH2O) to reduce the SSA concentration to 0.5 % for the assay.

Plasma
1) Centrifuge an anticoagulant-treated blood at 1,000 xg for 10 minutes at 4ºC.
2) Transfer the top plasma layer to a new tube and add the half the volume of 5% SSA equivalent to the tube.
3) Centrifuge at 8,000 xg for 10 minutes at 4ºC.
4) Transfer the supernatant to a new tube and add ddH2O to reduce the SSA concentration to 0.5% for the assay.

Erythrocytes
1) Centrifuge an anticoagulant-treated blood at 1,000 xg for 10 minutes at 4ºC.
2) Discard the supernatant and the white buffy coat.
3) Add 4 times volume of 5% SSA to the erythrocytes to lysis.
4) Centrifuge at 8,000 xg for 10 minutes at 4ºC.
5) Transfer the supernatant to a new tube, and add ddH2O to reduce the SSA concentration to 0.5% for the assay.

Cells (1x107 cells)
1) Collect cells by centrifugation at 200 xg for 10 minutes at 4ºC. Discard the supernatant.
2) Wash the cells with 300 μl of PBS and centrifuge at 200 xg for 10 minutes at 4ºC. Discard the supernatant.
3) Add 80 μl of 10 mM HCl, and lyse cells by freezing and thawing twice.
4) Add 20 μl of 5% SSA and centrifuge at 8,000 xg for 10 minutes.
5) Transfer the supernatant to a new tube, and add ddH2O to reduce the SSA concentration to 0.5% for the assay.



Item # Description/Size Availability Qty Break Price Quantity
G257-10
200 tests
1-2 business days 1 $574.00

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