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| Product Name | Glutathione (GSH/GSSG) Ratio Detection Assay Kit |
| Catalog No. | CMTR-HMM-0058 |
| Description | A fluorometric assay for the separate quantification of reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG) in biological samples. The kit uses a thiol-reactive fluorescent probe combined with an enzymatic recycling method to measure total glutathione and GSSG separately, enabling calculation of the GSH/GSSG ratio as a cellular oxidative stress indicator. |
| Intended Use | Measuring intracellular glutathione levels and redox balance in cells and tissues for oxidative stress research, toxicological screening, drug metabolism studies, and evaluation of antioxidant defense capacity. |
| Principle / Technology | GSH is selectively detected by a thiol-reactive probe that generates a fluorescent product upon reaction with the sulfhydryl group; for total glutathione measurement, glutathione reductase recycles GSSG back to GSH in the presence of NADPH, and all glutathione is subsequently quantified; for GSSG-specific measurement, GSH is first quenched using a thiol-scavenging reagent, then remaining GSSG is reduced by glutathione reductase and measured; the GSH/GSSG ratio (typically 30–100:1 in healthy cells) reflects redox homeostasis. |
| Detection Method | Fluorescence measurement (Ex/Em: 390–410/510–520 nm or standard blue excitation settings) using a fluorescence microplate reader |
| Sample Type | Cultured cells (adherent and suspension), fresh or snap-frozen tissue homogenates, whole blood and red blood cell lysates, plasma and serum, isolated mitochondrial preparations |
| Performance Range / Specifications | GSH standard curve: 0.1–10 μM; GSSG standard curve: 0.05–5 μM; linear detection of GSH in 1,000–100,000 cell equivalents per well; GSH/GSSG ratio measurable in 10 μg total protein lysate |
| Sensitivity / LOD | Sensitivity: approximately 10 nM GSH and 5 nM GSSG in purified solution; detectable in lysates from approximately 500 cells per well for typical proliferating cell lines |
| Specificity | The thiol-reactive fluorescent probe is highly selective for GSH over other cellular thiols (cysteine, coenzyme A, protein cysteines) under the optimized reaction pH; GSSG quantification is achieved after selective GSH masking; glutathione reductase specifically reduces GSSG to GSH using NADPH |
| Reaction Conditions / Protocol | Harvest cells (1–5 × 10^6), wash with cold PBS; prepare two sample aliquots: one for total glutathione and one for GSSG measurement; for GSSG sample, add GSH Quencher and incubate 10 minutes at room temperature; for both samples, add fluorescence detection reagent containing probe, glutathione reductase, and NADPH; incubate 30–60 minutes at room temperature protected from light; measure fluorescence (Ex/Em 400/515 nm); calculate GSH = Total Glutathione - 2 × GSSG |
| Components / Formulation | GSH Standard (reduced L-glutathione, lyophilized), GSSG Standard (oxidized glutathione, lyophilized), Thiol Green Fluorescent Probe (in DMSO), Glutathione Reductase, NADPH (lyophilized), Glutathione Assay Buffer, GSH Quencher (thiol-scavenging reagent), Sulfosalicylic Acid (SSA) for sample deproteinization |
| Storage Conditions | All components at -20°C protected from light except Assay Buffer (2–8°C); thiol probe sensitive to moisture; reconstituted NADPH for fresh use only |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 100 assays, 400 assays (96-well format) |
| Product Form | Lyophilized standards and enzymes with liquid buffers and fluorescent probe |
| Quality Control | Each lot standardized against reference GSH and GSSG concentrations with linear standard curves (R2 ≥0.99); positive control cell lysate tested; thiol-probe lot-to-lot fluorescence intensity within 20% |
| Key Features | Dual measurement of both GSH and GSSG from the same cell lysate; enzymatic recycling method amplifies signal for picomolar sensitivity; robust assay applicable to diverse biological matrices; provides the GSH/GSSG ratio used as a hallmark of oxidative stress |
For research use only, not for clinical use.
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