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| Product Name | Glucose-6-Phosphate Dehydrogenase Activity Colorimetric Assay Kit |
| Catalog No. | CMTR-HMM-0065 |
| Description | A colorimetric assay for the quantitative measurement of glucose-6-phosphate dehydrogenase enzymatic activity in biological samples. G6PD catalyzes the first committed step of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphogluconolactone with parallel reduction of NADP+ to NADPH, which is detected through a coupled reaction producing a colored formazan. |
| Intended Use | Measuring G6PD activity for the study of pentose phosphate pathway metabolism, NADPH generation for anabolic biosynthesis and redox homeostasis, and screening for G6PD deficiency in erythrocyte populations. |
| Principle / Technology | G6PD catalyzes the oxidation of glucose-6-phosphate to 6-phosphoglucono-δ-lactone with concomitant reduction of NADP+ to NADPH; in the assay, the generated NADPH reduces a tetrazolium dye (WST-8) to a colored formazan product in the presence of an electron mediator, providing colorimetric detection at 450 nm proportional to G6PD enzyme activity. |
| Detection Method | Colorimetric absorbance measurement at 450 nm using a microplate spectrophotometer with optional kinetic reading mode |
| Sample Type | Cell lysates from cultured mammalian cells, erythrocyte lysates, tissue homogenates (liver, adipose, adrenal gland), purified recombinant human G6PD, yeast and bacterial extracts |
| Performance Range / Specifications | NADPH standard curve: 0.5–20 nmol/well; G6PD activity detectable 0.1–20 mU/well; incubation 10–60 minutes at 37°C depending on activity; one unit enzyme produces 1 μmol NADPH per minute |
| Sensitivity / LOD | Detection limit: approximately 0.02 mU/well purified G6PD; measurable in lysates from approximately 1,000 cells per well for metabolically active cell types |
| Specificity | G6PD is highly specific for glucose-6-phosphate as its substrate; 6-phosphogluconate dehydrogenase (6PGD), the next pentose phosphate pathway enzyme, does not utilize G6P as substrate; NADP+ serves as the exclusive coenzyme; NAD+ is not utilized at detectable levels under assay conditions |
| Reaction Conditions / Protocol | Prepare cell or tissue lysates in cold G6PD Assay Buffer containing 1% Triton X-100 and protease inhibitors; centrifuge 10,000 × g for 10 minutes at 4°C; collect supernatant; add 50 μL diluted sample to microplate; add 50 μL Reaction Mix (G6P substrate, NADP+, WST-8 tetrazolium, electron mediator in assay buffer); measure absorbance at 450 nm kinetically at 37°C for 20–60 minutes, or as endpoint with stopping reagent; prepare NADPH standard curve for quantification of product formed |
| Components / Formulation | G6PD Assay Buffer (Tris-HCl, pH 8.0, with MgCl2), Glucose-6-phosphate Substrate (200 mM stock), NADP+ (15 mM stock), WST-8 Solution (tetrazolium dye), Diaphorase/Electron Mediator Mix, NADPH Standard (for calibration), G6PD Positive Control (purified Leuconostoc mesenteroides enzyme, lyophilized), Stop Solution |
| Storage Conditions | All components at -20°C protected from light except Assay Buffer (2–8°C); reconstituted NADPH standard single-use only; enzyme and substrate solutions stable 4 weeks at -20°C |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 100 assays, 500 assays (96-well format) |
| Product Form | Lyophilized enzyme standard and liquid concentrated substrate/coenzyme/dye reagents |
| Quality Control | Each lot tested for linear NADPH standard curve (R2 ≥0.995); G6PD positive control specific activity within specified range; reaction blank (no enzyme) absorbance drift <0.01 OD per hour |
| Key Features | Colorimetric detection compatible with standard absorbance plate readers; includes G6PD positive control for assay validation; kinetic and endpoint modes available; applicable to erythrocyte G6PD deficiency screening with modified protocol using whole blood lysates |
For research use only, not for clinical use.
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