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| Product Name | Glutathione Peroxidase (GPx) Activity Colorimetric Assay Kit |
| Catalog No. | ETR-HMM-0068 |
| Description | A colorimetric assay for quantifying total glutathione peroxidase activity using tert-butyl hydroperoxide as the organic peroxide substrate. GPx reduces the peroxide to an alcohol using glutathione (GSH) as the electron donor; the resulting GSSG is recycled back to GSH by glutathione reductase with NADPH oxidation, measured at 340 nm. |
| Intended Use | Quantitative measurement of cellular antioxidant GPx enzyme activity in studies of oxidative stress defense, selenium metabolism, cancer biology, and assessment of antioxidant enzyme status in aging and inflammation research. |
| Principle / Technology | GPx (EC 1.11.1.9) catalyzes: 2 GSH + ROOH → GSSG + ROH + H2O; the enzyme uses reduced glutathione (GSH) as a cofactor and reduces organic peroxides or H2O2; GSSG generated is continuously recycled to GSH by glutathione reductase using NADPH; the total GPx assay couples GPx activity to glutathione reductase in an indirect linked kinetic assay where NADPH oxidation (A340 decrease) is stoichiometrically proportional to GPx activity. |
| Detection Method | UV kinetic absorbance measurement at 340 nm using a UV-capable spectrophotometer or microplate reader |
| Sample Type | Cell lysates from cultured mammalian cells (hepatocytes, erythrocytes, cardiac cells are GPx-rich), tissue homogenates (liver, kidney, heart, lung), erythrocyte lysates, plasma (for plasma GPx3 isoform) |
| Performance Range / Specifications | GPx activity range: 0.1–20 nmol NADPH oxidized/min per μg protein; one unit = amount oxidizing 1 μmol NADPH per minute at 25°C; substrate (tBHP) concentration 0.2 mM; linear kinetic rate over 5 minutes |
| Sensitivity / LOD | Lower detection limit: approximately 0.005 U/mL purified GPx; detectable from approximately 20 μg total protein lysate for selenium-replete cells |
| Specificity | Coupled glutathione reductase recycling assay measures total GPx activity of all isoforms; selenium-dependent GPx (GPx1, GPx2, GPx3, GPx4) and phospholipid hydroperoxide GPx activities are included; non-selenium-dependent GST (glutathione S-transferase) activity toward tBHP may contribute 5–10% background, subtracted using N-ethylmaleimide (NEM) to quench glutathione-dependent background |
| Reaction Conditions / Protocol | Prepare cell lysate in Assay Buffer; add 20 μL diluted lysate to microplate; add 160 μL Reaction Mix (GSH, NADPH, glutathione reductase, assay buffer); allow 5 minute pre-reaction at room temperature; add 20 μL tBHP Substrate Solution to initiate; immediately begin kinetic reading at 340 nm every 30 seconds for 5 minutes; calculate mean ΔA340/min from linear kinetic portion; one GPx unit = nmol NADPH oxidized per minute |
| Components / Formulation | Reaction Buffer (Tris-HCl pH 7.6, EDTA, KCl, NaN3), Glutathione Reductase (lyophilized, 5 U/vial), NADPH (lyophilized), Reduced Glutathione (GSH, lyophilized), tert-Butyl Hydroperoxide Substrate (5 mM stock), GPx Positive Control (bovine erythrocyte GPx, lyophilized), N-Ethylmaleimide (NEM, background control) |
| Storage Conditions | All lyophilized components at -20°C protected from light; NADPH and GSH highly sensitive to oxidation, prepare fresh; glutathione reductase at -20°C in glycerol storage; tBHP at 2–8°C (avoid inhalation) |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 100 assays, 400 assays (96-well UV format) |
| Product Form | Multiple lyophilized enzyme and coenzyme components with liquid substrate and buffer |
| Quality Control | Each lot validated with bovine erythrocyte GPx reference standard; specific activity within ±20% of reference; NEM background subtraction efficiency confirmed; NADPH standard curve for rate calculation included in protocol |
| Key Features | Coupled glutathione reductase recycling method for continuous kinetic monitoring; total selenium-dependent and non-selenium-dependent GPx measured; NEM control allows background estimation from non-GPx glutathione-dependent reactions; applicable to all mammalian species due to conserved GPx mechanism |
For research use only, not for clinical use.
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