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| Product Name | Triglyceride Quantification Fluorometric Assay Kit, Lipase-Free |
| Catalog No. | CMTR-HMM-0077 |
| Description | Lipase-free fluorometric assay kit for quantitative measurement of triglyceride content in cell lysates, tissue homogenates, serum, and plasma. The assay employs a coupled enzymatic reaction: lipase first hydrolyzes triglycerides to glycerol and free fatty acids; glycerol kinase then phosphorylates glycerol with ATP to glycerol-3-phosphate; glycerol-3-phosphate oxidase converts this to dihydroxyacetone phosphate and hydrogen peroxide; finally, horseradish peroxidase uses H2O2 to oxidize a fluorogenic probe (ADHP/Amplex Red analog) to fluorescent resorufin (Ex/Em 535/587 nm). Lipase is provided separately, enabling detection of free glycerol background in a parallel reaction to calculate true triglyceride content by subtraction. |
| Intended Use | Quantitative measurement of intracellular and extracellular triglyceride levels for metabolic disease research, adipogenesis and lipogenesis studies, steatosis assessment, and metabolic drug screening in cell and animal models. |
| Principle / Technology | Enzymatic triglyceride hydrolysis coupled to glycerol phosphorylation, oxidation and fluorogenic probe detection; parallel free glycerol measurement (without lipase) enables background subtraction; fluorescence proportional to triglyceride concentration. |
| Detection Method | Fluorescence microplate reader (Ex/Em 535/590 nm); endpoint reading after 30-60 minutes at 37 °C. |
| Sample Type | Cell lysates (adipocytes, hepatocytes, myocytes); liver, muscle, and adipose tissue homogenates; serum and plasma (EDTA or heparin); cell culture medium for lipolysis assays. |
| Performance Range / Specifications | Linear range: 0.2-20 µM triglyceride (as triolein equivalent); free glycerol linear range: 0.1-10 µM; applicable to 2-200 pmol per well. |
| Sensitivity / LOD | Detection limit: 0.05 µM triglyceride (5 pmol in 100 µL); signal differentiation of 0.2 µM from blank. |
| Specificity | Glycerol kinase specifically phosphorylates glycerol; triglyceride-specific after free glycerol subtraction; minimal interference from mono- and diglycerides, phospholipids, or cholesterol esters. |
| Reaction Conditions / Protocol | Prepare sample + assay buffer; add lipase (for total glycerol) or buffer (for free glycerol); incubate 20 min at 37 °C; add enzyme-probe mixture; incubate 30-60 min at 37 °C; measure fluorescence. |
| Components / Formulation | Lipase (from Pseudomonas sp.), glycerol kinase, glycerol-3-phosphate oxidase, HRP, fluorogenic probe (ADHP), triglyceride standard (triolein, 1 mM), glycerol standard (1 mM), ATP, assay buffer, 96-well black plate. |
| Storage Conditions | Store enzymes and probe at -20 °C desiccated; standards at -20 °C; buffer at 2-8 °C. |
| Shelf Life | 6 months from date of manufacture. |
| Package Specifications | 100 tests, 400 tests (96-well format). |
| Product Form | Lyophilized enzymes and probe; liquid standards in ethanol. |
| Quality Control | Each lot tested for standard curve linearity (R² >0.995); free glycerol background in standard <0.5% of triglyceride value; spike recovery 90-110%. |
| Key Features | Lipase-free parallel free glycerol measurement; fluorometric high sensitivity; separate triglyceride and glycerol quantification; triolein-based standard; adipose and liver tissue compatible. |
| Purity | Triolein standard ≥99%; glycerol standard ≥99%; probe ≥98%; enzymes recombinant and purified. |
| Concentration | Triolein and glycerol standards: 1 mM stocks; working concentrations per protocol. |
| Activity / Unit Definition | Lipase: 1 U hydrolyzes 1 µmol triglyceride per minute; HRP: RZ >3.0. |
| Molecular Weight | Triolein: 885.43 g/mol (C57H104O6); glycerol: 92.09 g/mol. |
| Source / Origin | Recombinant enzymes expressed in E. coli; synthetic fluorogenic probe; purified triolein from olive oil. |
| pH Range / Optimal pH | Assay buffer pH 7.4-7.6. |
| Shipping Conditions | Cold pack (enzymes and probe on dry ice). |
| Expiration Date / Stability | 6 months at recommended storage; reconstituted enzymes stable 1 month at 2-8 °C. |
| Regulatory / Compliance | For research use only; not for diagnostic applications. |
| Compatibility | Compatible with cell and tissue lysates in non-ionic detergent buffers. Triton X-100 and NP-40 up to 0.1% are tolerated. SDS and ionic detergents inhibit enzyme cascade — use recommended homogenization buffer. |
| Recommended Buffer System | HEPES buffer with MgCl2 and ATP, pH 7.5. |
| Application Notes / Precautions | Include lipase-free control wells for each sample to measure endogenous free glycerol. Homogenize tissues in chloroform:methanol (2:1) for complete lipid extraction if using solid tissues. Normalize triglyceride to protein content for cell/tissue samples. Serum samples with high glycerol (e.g., from contaminated blood collection tubes with glycerol-lubricated stoppers) will give falsely elevated results — use only glycerol-free collection tubes. |
| Batch-to-Batch Consistency | Triolein standard concentration verified by gravimetric analysis; enzyme cascade activity within ±15% of reference lot. |
For research use only, not for clinical use.
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