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| Product Name | Free Glycerol Colorimetric Assay Kit, Glycerol Kinase-Phosphate Method |
| Catalog No. | CMTR-HMM-0078 |
| Description | Colorimetric assay kit for quantitative determination of free glycerol levels in biological samples using the glycerol kinase-glycerol phosphate oxidase coupled enzymatic method. Glycerol is phosphorylated by glycerol kinase and ATP to glycerol-1-phosphate, which is then oxidized by glycerol phosphate oxidase to dihydroxyacetone phosphate with concomitant production of hydrogen peroxide. Peroxidase catalyzes the H2O2-dependent oxidative coupling of 4-aminoantipyrine (4-AAP) with a phenolic chromogen to produce a quinoneimine dye with absorbance maximum at 540 nm. The color intensity is directly proportional to glycerol concentration in the sample. |
| Intended Use | Quantitative measurement of free glycerol in serum, plasma, cell culture medium, and tissue homogenates as a marker of lipolysis activity, triglyceride metabolism, and metabolic status in diabetes, obesity, and metabolic syndrome research. |
| Principle / Technology | Glycerol kinase → glycerol-1-phosphate; glycerol phosphate oxidase → H2O2; peroxidase → quinoneimine dye (absorbance 540 nm); reaction is linear with glycerol concentration; compatible with automated analyzers and manual microplate formats. |
| Detection Method | Colorimetric microplate reader or spectrophotometer at 540 nm; endpoint reading after 15-30 minutes at 37 °C. |
| Sample Type | Serum, EDTA or heparin plasma, cell culture supernatant, tissue homogenates, adipocyte-conditioned medium. |
| Performance Range / Specifications | Linear range: 5-500 µM glycerol (0.05-5 mg/dL); applicable to 0.5-50 nmol per well; correlation with reference GC-MS method R² >0.98. |
| Sensitivity / LOD | Detection limit: 2 µM glycerol (0.2 nmol/well); absorbance change >0.01 AU at detection limit. |
| Specificity | Glycerol kinase highly specific for glycerol; <0.1% cross-reactivity with ethylene glycol, propylene glycol, dihydroxyacetone, glyceraldehyde; minimal interference from glucose, lactate, or pyruvate at physiological concentrations. |
| Reaction Conditions / Protocol | Mix sample with enzyme-chromogen reagent; incubate 15-30 min at 37 °C; read absorbance at 540 nm; calculate concentration from glycerol standard curve. |
| Components / Formulation | Glycerol kinase, glycerol phosphate oxidase, peroxidase, 4-aminoantipyrine (4-AAP), phenolic chromogen, ATP, glycerol standard (500 µM), assay buffer, detailed protocol. |
| Storage Conditions | Store at 2-8 °C; protect from light. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 100 mL total reagent (sufficient for ~500 tests in 200 µL format), 500 mL bulk reagent. |
| Product Form | Lyophilized or liquid-stable two-component reagent; reconstitute as directed. |
| Quality Control | Each lot tested for linearity (R² >0.995), sensitivity, and glycerol recovery (95-105%) in spiked serum samples. |
| Key Features | Simple colorimetric detection at 540 nm; glycerol kinase-phosphatase coupled method; linear to 500 µM; compatible with automated analyzers; minimal sample preparation; long liquid stability after reconstitution. |
| Purity | 4-AAP ≥98%; glycerol ≥99.5% standard; enzymes purified to homogeneity. |
| Concentration | Glycerol standard: 500 µM; prepare serial dilutions for standard curve. |
| Activity / Unit Definition | Glycerol kinase: ≥50 U/mL reagent; glycerol phosphate oxidase: ≥20 U/mL; peroxidase: ≥10 U/mL. |
| Molecular Weight | Glycerol: 92.09 g/mol. |
| Source / Origin | Glycerol kinase from E. coli (recombinant); glycerol phosphate oxidase from Pediococcus sp.; peroxidase from horseradish. |
| pH Range / Optimal pH | Assay buffer pH 7.0-7.5; optimal enzyme activity at pH 7.2. |
| Shipping Conditions | Cold pack (2-8 °C) for liquid reagent; ambient for lyophilized. |
| Expiration Date / Stability | 12 months at 2-8 °C; reconstituted reagent stable 30 days at 2-8 °C or 3 days at room temperature. |
| Regulatory / Compliance | For research and in vitro diagnostic use; manufactured under ISO 13485 quality system. |
| Compatibility | Compatible with serum, plasma, and cell culture media. Hemolysis (Hb >200 mg/dL) causes positive interference; icterus (bilirubin >20 mg/dL) may cause spectral interference; lipemic samples should be ultracentrifuged before assay. |
| Recommended Buffer System | PIPES buffer with MgCl2 and ATP, pH 7.2. |
| Application Notes / Precautions | Use glycerol-free tubes and pipette tips for sample collection and handling. Avoid glycerol-containing hand lotions and soaps as they are a common source of contamination. For cell culture supernatant, centrifuge to remove cell debris. Serum separator tubes with glycerol-lubricated stoppers should not be used. Include glycerol-free water blank with each run. |
| Batch-to-Batch Consistency | Glycerol standard concentration within ±3% of specification; reagent absorbance blank <0.05 AU at 540 nm. |
For research use only, not for clinical use.
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