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| Product Name | Ammonia/Ammonium Fluorometric Assay Kit, Glutamate Dehydrogenase Method |
| Catalog No. | CMTR-HMM-0079 |
| Description | Fluorometric assay for quantitative measurement of ammonia (NH3) and ammonium ion (NH4+) in cell lysates, tissue homogenates, culture media, and biological fluids. The assay uses glutamate dehydrogenase (GDH) to catalyze the reductive amination of α-ketoglutarate with ammonium and NADH to form glutamate and NAD+. The decrease in NADH fluorescence (Ex/Em 340/440 nm) or absorbance (340 nm) is proportional to the ammonium concentration in the sample. A deproteinization step removes endogenous enzymes and proteins that may interfere with NADH measurement, while provided standards enable precise quantification. |
| Intended Use | Quantitative measurement of ammonia/ammonium levels as a marker of amino acid catabolism, ureagenesis, glutamine metabolism, and nitrogen balance in hepatocyte function, cancer metabolism, and inborn errors of metabolism research. |
| Principle / Technology | GDH-catalyzed reaction: NH4+ + α-ketoglutarate + NADH → glutamate + NAD+ + H2O; decrease in NADH absorbance (340 nm) or fluorescence (Ex 340/Em 440 nm) proportional to NH4+; specificity ensured by GDH substrate selectivity. |
| Detection Method | Fluorescence (Ex/Em 340/440 nm) or absorbance (340 nm) microplate reader; endpoint or kinetic mode. |
| Sample Type | Cell lysates, tissue homogenates, plasma, serum, urine, cell culture supernatant. |
| Performance Range / Specifications | Linear range: 1-100 µM NH4+ (fluorescence) or 5-200 µM (absorbance); applicable to 0.1-10 nmol per well. |
| Sensitivity / LOD | Detection limit: 0.5 µM NH4+ (fluorescence mode); 2 µM (absorbance mode). |
| Specificity | GDH is specific for NH4+; <0.1% cross-reactivity with glutamine, asparagine, urea, amino acids, or amines. |
| Reaction Conditions / Protocol | Deproteinize sample (optional for cell culture medium); add GDH/α-KG/NADH reagent; incubate 30 min at 37 °C; measure fluorescence or absorbance decrease; compare to NH4Cl standard curve. |
| Components / Formulation | Glutamate dehydrogenase (lyophilized), α-ketoglutarate, NADH (lyophilized), ammonium chloride standard (10 mM), deproteinizing reagent (TCA), neutralizing buffer, assay buffer. |
| Storage Conditions | Store GDH and NADH at -20 °C; other reagents at 2-8 °C. |
| Shelf Life | 6 months from date of manufacture. |
| Package Specifications | 100 tests, 400 tests. |
| Product Form | Lyophilized enzymes and cofactors; liquid standards and buffers. |
| Quality Control | Each lot tested for standard curve linearity (R² >0.99), enzymatic activity, and ammonium recovery (90-110%) in spiked serum. |
| Key Features | Enzymatic GDH method; dual-read mode (fluorescence or absorbance); deproteinization step included; NADH monitoring; high specificity for NH4+. |
| Purity | GDH specific activity ≥50 U/mg; NADH ≥97% purity; α-ketoglutarate ≥99%. |
| Concentration | NH4Cl standard: 10 mM stock; dilute to 100 µM working standard. |
| Activity / Unit Definition | GDH: 1 unit converts 1 µmol α-ketoglutarate to glutamate per minute at pH 8.0, 25 °C. |
| Molecular Weight | NH4Cl: 53.49 g/mol; NADH: 709.43 g/mol; α-ketoglutarate: 146.10 g/mol. |
| Source / Origin | GDH from bovine liver or recombinant; synthetic cofactors and substrates. |
| pH Range / Optimal pH | Optimal GDH activity at pH 8.0; assay buffer pH 7.8-8.2. |
| Shipping Conditions | Cold pack (GDH and NADH on dry ice). |
| Expiration Date / Stability | 6 months at -20 °C; reconstituted GDH stable 2 weeks at 2-8 °C. |
| Regulatory / Compliance | For research use only; not for diagnostic procedures. |
| Compatibility | Compatible with deproteinized samples; TCA deproteinization removes endogenous enzymes. Ammonia-free water must be used for all reagent preparation. Glassware should be acid-washed to remove trace ammonia. |
| Recommended Buffer System | Triethanolamine buffer with EDTA, pH 8.0. |
| Application Notes / Precautions | All water and buffers must be ammonia-free — use freshly deionized water or purchase ammonia-free water. Avoid ammonium contamination from laboratory air (ammonia-based cleaning products, tobacco smoke). Prepare samples quickly and keep sealed as blood/serum ammonia increases at room temperature (~0.5 µM/hour). For plasma ammonia, collect in EDTA tubes, place immediately on ice, and assay within 30 minutes. |
| Batch-to-Batch Consistency | GDH enzymatic activity within ±15% of reference lot; NH4Cl standard concentration verified by ion-selective electrode. |
For research use only, not for clinical use.
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