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GSSG/GSH Quantification Kit

Item # Unit Size
G257-10
200 tests

For Research Use Only Products

  • Selectively Quantify Glutathione (Oxidized form and/or Reduced form)
  • Contains Masking Reagent of GSH
  • Easy and Simple procedure

MSDS

Storage Condition : 0-5oC, protect from light
Shipping Condition :  blue ice

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, only the GSSG is detected by measuring the absorption (λmax = 412 nm) of DTNB (5,5’-dithiobis (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

Fig. 1 Mechanism of total glutathione quantification


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

     
Preparation of 5% 5-Sulfosalicylic Acid (SSA) Solution
Note: SSA is not included in this kit.
1. Dissolve 1 g SSA in 19 ml water.
2. Store the solution at 4°C (stable for 6 months at 4°C).

Preparation of Sample Solution

Cells (Adhesive cells: 5x105 cells; Leukocyte cells: 1x106 cells)
1. Collect cells by centrifugation at 200 g for 10 min at 4°C. Discard the supernatant.
2. Wash the cells with 300 μl PBS and centrifuge at 200 g for 10 min at 4°C. Discard the supernatant.
3. Add 80 μl 10 mM HCl, and lyse the cells by freezing and thawing twice.
4. Add 20 μl 5% SSA and centrifuge at 8,000 g for 10 min.
5. Transfer the supernatant to a new tube, and use it for the assay. If the final concentration of SSA is over 1%, add ddH2O to reduce the concentration of SSA from 0.5 to 1%.

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

Plasma
1. Centrifuge anticoagulant-treated blood at 1,000 g for 10 min at 4°C.
2. Transfer the top plasma layer to a new tube and add 5% SSA equivalent to half of the volume of the plasma.
3. Centrifuge at 8,000 g for 10 min at 4°C
4. Transfer the supernatant to a new tube, and add ddH2O to reduce the concentration of SSA from 0.5 to 1%. Use it for the assay.

Erythrocytes
1. Centrifuge anticoagulant-treated blood at 1,000 g for 10 min at 4°C.
2. Discard the supernatant and the white buffy layer.
3. Lyse the erythrocytes with 5% SSA equivalent to 4 times the volume of the erythrocytes.
4. Centrifuge at 8,000 g for 10 min at 4°C.
5. Transfer the supernatant to a new tube, and add ddH2O to reduce the concentration of SSA from 0.5 to 1%. Use it for the assay. Erythrocytes can be isolated from the remaining sample solution after the plasma sample isolation.

Preparation of Assay Solutions

Substrate Working Solution
Add 1 ml Buffer Solution to 1 vial of Substrate, and dissolve. Substrate working solution is stable for 2 months at -20°C.
Enzyme Working Solution
Mix Enzyme solution with pipetting before using. Take out 20 μl Enzyme solution and mix it with 4 ml Buffer solution. Enzyme working solution is stable for 2 months at 4°C.
Coenzyme Working Solution
Add 0.7 ml ddH2O to the Coenzyme vial and dissolve. If you don’t use all of the coenzyme working solution in one day, aliquot it into microtubes and store at -20oC. If you use all of the coenzyme working solution in one day, just add 6.3 ml Buffer solution to the vial.
The Coenzyme vial is under vacuum pressure; carefully open the cap or use a syringe to add Buffer solution. Since the Coenzyme working solution dissolved in the Buffer solution is not stable, use it in one day. The coenzyme solution prepared with ddH2O is only stable for 2 months at -20oC. Dilute 10 times with Buffer solution to prepare Working solution prior to use.

GSH Standard Solutions
To prepare 200 μM GSH standard solution, add 2 ml of 0.5-1% SSA to the Standard GSH vial and dissolve. Dilute 100 μl of the 200 μM GSH standard solution with 100 μl of 0.5% SSA, and repeat using serial dilution to prepare the following GSH standard solutions:
100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM and 0.
The Standard GSH vial is under vacuum pressure; carefully open the cap or use a syringe to add SSA. GSH powder is difficult to see. The GSH standard solutions are stable for 2 months at -20°C.

Total Glutathione Detection - Standard Method

Detection Range: 5-100 μM
1. To each well, add 20 μl of Enzyme working solution, 140 μl of Coenzyme working solution, and 20 μl of either one of the GSH standard solutions or the sample solution.a)
2. Incubate the plate at 37°C for 10 min.
3. Add 20 μl of Substrate working solution, and incubate the plate at 37°C for 5-10 min.
4. Read the absorbance at 405 nm or 415 nm using a microplate reader.
5. Determine the concentration of GSH in the sample solution using a calibration curveb).

a) Adjust the concentration of SSA in the sample solution to 0.5-1% with ddH2O before the assay. High concentrations of SSA (>1 %) interfere with the assay.
b) Since the colorimetric reaction is stable and the O.D. increases linearly over 30 min, GSH concentration can be determined by kinetic or pseudo-endpoint (no stopping reaction, quick measurement of O.D. at certain time periods between 5 and 10 min) methods.


Total Glutathione Detection - High Sensitivity Method Detection Range: 0.5-25 μM
1. To each well, add 20 μl of Enzyme working solution, 140 μl of Coenzyme working solution, and 20 μl of either one of the GSH standard solutionsa) or the sample solutionb).
2. Incubate the plate at 30°C for 10 min.
3. Add 20 μl of Substrate working solution, and incubate the plate at 37°C for 20-40 min.
4. Read the absorbance at 405 nm or 415 nm using a microplate reader.
5. Determine the concentration of GSH in the sample solution using a calibration curve.

a) Prepare 50 mM GSH standard solution, and then prepare different concentrations of GSH standard solutions by serial dilution with 0.5% SSA as follows: 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM, 0.39 μM and 0.
b) Adjust the concentration of SSA in the sample solution to 0.5-1% with ddH2O before the assay. Higher concentrations of SSA (>1%) interfere with the assay.


Determination of Total Glutathione (GSH and GSSG) Concentration
Determine the total glutathione concentration in the sample solution using the following equations. Since the values obtained by these equations are the amount of total glutathione in treated sample solutions, further calculations are necessary if the actual concentration of glutathione in cells or tissues needs to be determined.


References
G. L. Ellman, Arch. Biochem. Biophys., 82, 70 (1959);
O. W. Griffith, Anal. Biochem., 106, 207 (1980);
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M. A. Baker, et al., Anal. Biochem., 190, 360 (1990);
C. Vandeputte, et al., Cell Biol. Toxicol., 10, 415 (1994);
S. A. McGrath-Morrow, et al., Am. J. Respir. Cell Mol. Biol., 27, 99 (2002);
R. M. Tuder, et al., Am. J. Respir. Cell Mol. Biol., 29, 88 (2003);
J. Zielinska-Park, et al., Carcinogenesis, 25, 1727 (2004);
X. Cui, et al., Toxicol. Sci., 82, 478 (2004);
K. Imai, et al., J. Biol. Chem., 280, 26701 (2005);
L. Wang, et al., J. Biol. Chem., 281, 24553 (2006);
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M. L. Mulbern, et al., Invest. Ophthalmol. Vis. Sci., 47, 3951 (2006);
S. Kasagi, et al., Am. J. Physiol. Lung Cell Mol. Physiol., 290, L396 (2006);
T. Sato, et al., Am. J. Respir. Crit. Care Med., 174, 530 (2006);
K. Yoh, et al., Genes Cells., 13, 1150 (2008);
Y. Keum, et al., Carcinogenesis, 29, 594 (2008);
J. R. Ridpath, et al., Cancer Res., 67, 11117 (2007).
Literature used "GSSG/GSH Quantification Kit"

N. Miura, Y. Yanagiba, K. Ohtani, M. Mita, M. Togawa, and T. Hasegawa, Diurnal Variation of cadmium-induced mortality in mice, 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.