BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
Store at 2-8°C,Protected from light,Store at -20°C Ships Ice chest + Ice pads Check lot-specific COA for exact specifications.
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Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Glutathione is a tripeptide containing a γ-amide bond and a sulfhydryl group, composed of glutamate, cysteine, and glycine. It is widely found in animal and plant tissues and microorganisms. In living organisms, it helps maintain normal immune system function and has antioxidant and detoxifying effects. Glutathione exists in two forms: reduced (GSH) and oxidized (GSSG). GSSG, also known as glutathione disulfide, is formed by the oxidation of two glutathione molecules. GSSG can be reduced back to GSH by glutathione reductase; therefore, it exists primarily in the reduced form in organisms. The ratio of reduced to oxidized glutathione (GSH/GSSG) serves as a key dynamic indicator for assessing the cellular redox state.
Detection Principle: Endogenous GSH in the sample is masked by 2-vinylpyridine. Under the catalysis of glutathione reductase (GR), GSSG is reduced to GSH. The generated GSH then reacts with 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) to produce yellow-colored 5-thio-2-nitrobenzoic acid (TNB), which has a characteristic absorption peak at 412 nm. The GSSG content is quantified by measuring the change in absorbance.
Detection Range: 1-20 µM
Sensitivity: 1 µM
Applicable Samples: Animal/plant tissues, blood cells, cells, bacteria, serum (plasma).
| O1492795 | Component | 96T | Storage |
| O1492795A | Extraction Buffer | 70 mL×2 | 2-8℃ |
| O1492795B | Inhibitor | 210 μL | -20℃. Store in the dark. |
| O1492795C | Assay Buffer | 20 mL | 2-8℃ |
| O1492795D | GR | 14 μL | 2-8℃. Store in the dark. |
| O1492795E | GR Cofactor | 2 EA | -20℃. Store in the dark. |
| O1492795F | Chromogen | 2 EA | 2-8℃. Store in the dark. |
O1492795G | Standard | 1 EA | 2-8℃. Store in the dark. |
User-Provided Instruments and Reagents
| Type | Name | Notes |
| Instrument | Microplate Reader | Capable of measuring absorbance at 412 nm. |
| Consumables | 96-well Microplate | Standard transparent plate. |
| Reagents | PBS / Deionized Water | For washing samples / Preparing reagents. |
| Others | Homogenizer (for tissue samples), water bath, ice bucket, low-temperature centrifuge, adjustable pipettes and tips | Using a multichannel pipette for large-scale detection can improve efficiency. |
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Reagent Preparation | Precautions |
| Extraction Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Diluted Extraction Buffer | Add 500 µL Extraction Buffer to 4.5 mL deionized water. | Obtained by 10-fold dilution of Extraction Buffer. |
| Inhibitor | Ready-to-use; equilibrate to room temperature before use. | Store at -20°C protected from light. Toxic and irritant; recommended to handle in a fume hood. |
| Assay Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| GR Dilution | Before use, prepare by adding 1 µL GR to 20 µL deionized water per sample. | Prepare freshly before use. |
| GR Cofactor Dilution | Before use, add 1.5 mL deionized water to each vial; equilibrate to room temperature protected from light. | After dissolution, store at -20°C protected from light for up to 1 month. |
| Chromogen Dilution | Before use, add 1.5 mL deionized water to each vial; equilibrate to room temperature protected from light. | After dissolution, store at 4°C protected from light for up to 1 month. |
| GSSG Standard | Dissolve in 1 mL of Diluted Extraction Buffer. | 20 mM; After dissolution, aliquot and store at -20°C protected from light for up to 1 month. |
2. Standard Preparation
Take 100 µL of the 20 mM GSSG standard and dilute with 900 µL Diluted Extraction Buffer to obtain a 2 mM GSSG standard solution.
Take 10 µL of the 2 mM GSSG standard and dilute with 990 µL Diluted Extraction Buffer to obtain a 20 µM GSSG standard solution.
Further dilute the standard as shown in the table below. A standard curve must be prepared for each experiment. Diluted standard solutions are unstable and must be used within 4 hours.
| Standard Working Solution | 20µM Standard (µL) | Diluted Extraction Buffer (µL) | Concentration (µM) |
| 1 | 100 | 0 | 20 |
| 2 | 80 | 20 | 16 |
| 3 | 60 | 40 | 12 |
| 4 | 40 | 60 | 8 |
| 5 | 20 | 80 | 4 |
| 6 | 10 | 90 | 2 |
| 7 | 5 | 95 | 1 |
3. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 10 days. Because the Extraction Buffer contains a protein precipitant, the supernatant cannot be used for protein concentration determination. If protein content needs to be measured, prepare another identical sample using deionized water instead of Extraction Buffer.
3.1 Animal/Plant Tissue Samples:
Use fresh tissue samples whenever possible. Weigh 0.1 g of tissue, add 1 mL of pre-cooled Extraction Buffer, and homogenize quickly on ice (pre-cool the homogenizer on ice). Centrifuge the homogenate at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
3.2 Serum/Plasma Samples:
Use fresh serum (plasma) whenever possible. Centrifuge the collected serum (plasma) at 600 g, 4°C for 10 min. Within 30 minutes, aspirate the supernatant into another tube. Add an equal volume of Extraction Buffer, mix, then centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
3.3 Cell or Bacterial Samples:
Use fresh cells (bacteria) whenever possible; avoid using frozen cells (bacteria). Collect 5×10⁶ cells (bacteria). Wash twice with 1 mL of pre-cooled PBS (resuspend in PBS, centrifuge at 600 g, 4°C for 10 min). Add 3 times the volume of Extraction Buffer relative to the cell (bacterial) pellet to resuspend the cells (bacteria). Disrupt by ultrasound on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
Note: Cells can also be extracted using a freeze-thaw method (not suitable for bacteria): Resuspend cells and subject to 2-3 rapid freeze-thaw cycles (freeze in liquid nitrogen, thaw in a 37°C water bath). Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
4. Assay Steps
4.1 Microplate Reader Preparation: Preheat for at least 30 minutes, set wavelength to 412 nm.
4.2 Assay System Setup (Step 1 - Pre-treatment): Perform the following operations in 1.5 mL EP tubes. This step must be done in EP tubes. Do not add Inhibitor directly to the 96-well plate as it may corrode the plate. Inhibitor is toxic and irritant; recommended to handle in a fume hood.
| Reagent | Blank Tube (µL) | Standard Tube (µL) | Test Tube (µL) |
| Sample | 0 | 0 | 3 |
| Deionized Water | 30 | 0 | 27 |
| Standard | 0 | 30 | 0 |
| Inhibitor | 1.5 | 1.5 | 1.5 |
4.3 Mix well and incubate at 37°C for 30 minutes. This becomes the "Mixture".
4.4 Assay System Setup (Step 2 - Reaction): Perform the following operations in a 96-well plate.
| Reagent | Blank Well (µL) | Standard Well (µL) | Test Well (µL) |
| Mixture | 21 | 21 | 21 |
| Assay Buffer | 140 | 140 | 140 |
| GR Dilution | 2 | 2 | 2 |
| GR Cofactor Dilution | 20 | 20 | 20 |
| Chromogen Dilution | 20 | 20 | 20 |
Precautions
1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.
2. The samples extracted with this kit are suitable for the detection of oxidized glutathione (GSSG). Because the extraction buffer contains a protein precipitant, the supernatant cannot be used for protein concentration determination. If protein content needs to be measured, prepare another identical sample using deionized water instead of the extraction buffer. For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.
3. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.
4. It is recommended to establish your own standard curve for improved accuracy. If not, you may refer to the typical standard curve formula provided in the results section for calculation.
5. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, hair cap, etc.) throughout the experiment and perform experiments in a fume hood or biosafety cabinet.
6. This product is for scientific research use only. Not intended for clinical diagnosis.
Frequently Asked Questions
Q: What should I do if the sample ΔA test is too high or too low?
Frequently Asked Questions Q: What should I do if the sample ΔA test is too high or too low? A: If the sample ΔA test is greater than the ΔA standard of the 20 µM standard, the GSSG content in the sample is too high. Dilute the sample appropriately with deionized water (multiply by the dilution factor in the calculation). If the sample ΔA test is less than 0.005, increase the sample amount.
Q: Can blood cell samples be detected?
A: Yes, blood cell samples can be detected. Centrifuge the collected anticoagulated blood at 600 g, 4°C for 10 min. Discard the upper plasma and wash the pellet 2-3 times with 3 volumes of PBS (resuspend blood cells in PBS, centrifuge at 600 g, 4°C for 10 min). Add an equal volume of Extraction Buffer, mix, and let stand at 4°C for 10 min. Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
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F2617580