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Total Glutathione (T-GSH)/Oxidized Glutathione (GSSG) Colorimetric Assay Kit
SKU: E-BC-K097-S-100
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Total Glutathione (T-GSH)/Oxidized Glutathione (GSSG) Colorimetric Assay Kit
| SKU # | E-BC-K097-S |
| Detection Instrument | Spectrophotometer (412 nm) |
| Detection Method | Colorimetric method |
Product Details
Properties
| Synonyms | T-GSH,GSSG |
| Sample Type | Serum, plasma, animal tissue, red blood cells, cultured cells |
| Sensitivity | 0.12 μmol/L T-GSH |
| Detection Range | 0.12-30 μmol/L T-GSH |
| Detection Method | Colorimetric method |
| Assay type | Quantitative |
| Assay time | 180 min |
| Precision | Average inter-assay CV: 4.700% | Average intra-assay CV: 0.900% |
| Other instruments required | Micropipettor, Incubator, Vortex mixer, Centrifuge |
| Other reagents required | PBS(0.01 M, pH 7.4), Absolute ethanol |
| Storage | Reagent 2, Reagent 3, Reagent 4, Reagent 8, Reagent 9: -20℃; others: 2-8℃ |
| Valid period | 12 months |
Dilution of Sample
It is recommended to take 2~3 samples with expected large difference to do pre-experiment before formal experiment and dilute the sample according to the result of the pre-experiment and the detection range (0.12-30 μmol/L T-GSH).
The recommended dilution factor for different samples is as follows (for reference only):
| Sample type | Dilution factor |
| Human serum | 1 |
| Rat plasma | 1 |
| Mouse serum | 1 |
| 10% Rat heart tissue homogenate | 10 |
| 10% Rat liver tissue homogenate | 60 |
| 10% Rat brain tissue homogenate | 10 |
Note: The diluent is reagent 3.
Detection Principle
GSSG is reduced to GSH by glutathione reductase, and GSH can react with DTNB to produce GSSG and yellow TNB. The amount of total glutathione (GSSG+GSH) determines the amount of yellow TNB. Thus the total glutathione can be calculated by measuring the OD value at 412 nm. The content of GSSG can be determined by first removing GSH from the sample with appropriate reagent and then using the above reaction principle.
Kit Components & Storage
| Item | Component |
Size 1 (50 assays) |
Size 2 (100 assays) |
Storage |
| Reagent 1 | Buffer Solution | 55 mL × 2 vials | 55 mL × 3 vials | 2-8℃, 12 months |
| Reagent 2 | GSSG Standard | 6.13 mg × 1 vial | 6.13 mg × 1 vial | -20℃, 12 month |
| Reagent 3 | Protein Precipitator | 60 mL × 1 vial | 60 mL ×2 vials | -20℃, 12 month |
| Reagent 4 | Enzyme Stock Solution | 77 μL × 1 vial | 154 μL × 1 vial | -20℃, 12 month |
| Reagent 5 | Chromogenic Agent | Powder × 1 vial | Powder × 2 vials | 2-8℃, 12 months, shading light |
| Reagent 6 | Diluent | 1.8 mL × 1 vial | 3.6 mL × 1 vial | 2-8℃, 12 months |
| Reagent 7 | GSH Scavenger Auxiliary Solution | 0.7 mL × 1 vial | 1.4 mL × 1 vial | 2-8℃, 12 months |
| Reagent 8 | GSH Scavenger | 0.1 mL × 1 vial | 0.2 mL × 1 vial | -20℃, 12 months, shading light |
| Reagent 9 | Substrate | Powder × 1 vial | Powder × 2 vials | -20℃, 12 months, shading light |
| Reagent 10 | Stop Solution | 25 mL × 1 vial | 50 mL × 1 vial | 2-8℃, 12 months |
Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.
Technical Data:
Parameter:
Intra-assay Precision
Three human serum samples were assayed in replicates of 20 to determine precision within an assay (CV = Coefficient of Variation).
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (μmol/L) | 3.50 | 16.40 | 23.50 |
| %CV | 1.2 | 0.9 | 0.6 |
Inter-assay Precision
Three human serum samples were assayed 20 times in duplicate by three operators to determine precision between assays.
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (μmol/L) | 3.50 | 16.40 | 23.50 |
| %CV | 4.5 | 4.7 | 4.9 |
Recovery
Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 97%.
| Sample 1 | Sample 2 | Sample 3 | |
| Expected Conc. (μmol/L) | 6.8 | 17.5 | 25 |
| Observed Conc. (μmol/L) | 6.7 | 16.8 | 24.0 |
| Recovery rate (%) | 99 | 96 | 96 |
Sensitivity
The analytical sensitivity of the assay is 0.12 μmol/L. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.
Standard Curve
As the OD value of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique or temperature effects), so the standard curve and data are provided as below for reference only:
| Concentration (μmol/L) | 0 | 1 | 2 | 5 | 8 | 10 | 15 |
| Average OD | 0.020 | 0.082 | 0.136 | 0.294 | 0.446 | 0.532 | 0.728 |
| Absoluted OD | 0 | 0.062 | 0.116 | 0.274 | 0.426 | 0.512 | 0.708 |