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| Product Name | ATP/ADP Ratio Bioluminescence Assay Kit |
| Catalog No. | CMTR-HMM-0056 |
| Description | A dual-reaction bioluminescence assay for sequential measurement of intracellular ADP and ATP levels from the same cell lysate. The ADP is first converted to ATP through an enzymatic reaction, and each ATP measurement is detected via luciferin-luciferase bioluminescence, enabling calculation of the ATP/ADP ratio as an indicator of cellular energy status. |
| Intended Use | Measurement of cellular energy charge and metabolic fitness in studies of mitochondrial dysfunction, drug-induced toxicity, ischemia-reperfusion injury, exercise physiology, and metabolic disorders. |
| Principle / Technology | After cell lysis, ATP is measured directly using firefly luciferase; then ADP in the same sample is enzymatically converted to ATP by pyruvate kinase in the presence of excess phosphoenolpyruvate (PEP); a second luciferase measurement detects total ATP (original ATP + converted ADP); the ADP concentration is calculated by subtraction, and the ATP/ADP ratio reflects cellular bioenergetic health. |
| Detection Method | Bioluminescence (relative light units) measured with a luminometer or multimode plate reader; sequential two-step protocol measuring ATP signal before and after ADP-to-ATP conversion |
| Sample Type | Mammalian cells (adherent and suspension), isolated mitochondria, tissue homogenates, blood and platelet samples, yeast and bacterial cultures |
| Performance Range / Specifications | ATP detection: 0.01–10 μM in lysate; ADP detection: 0.01–10 μM after conversion; ATP/ADP ratio >1 typically indicates healthy energetic status; luminescence signal half-life >30 minutes per measurement step |
| Sensitivity / LOD | Detection of ATP at approximately 0.1 pmol/well; detectable in lysates from as few as 100 cells per well for highly metabolic cell types |
| Specificity | Firefly luciferase exhibits absolute specificity for ATP as its nucleotide cofactor; GTP, CTP, UTP, and dATP produce negligible signals; ADP conversion by pyruvate kinase is highly specific with PEP as substrate; adenosine and AMP are not recognized by either reaction |
| Reaction Conditions / Protocol | Plate cells in opaque white-walled 96-well or 384-well plates; aspirate medium and add ATP Detection Reagent (lysis buffer + luciferin + luciferase); incubate 5 minutes at room temperature to stabilize luminescence; measure ATP signal; add ADP Converting Reagent (pyruvate kinase + PEP); incubate 5 minutes; measure total ATP signal; calculate ADP concentration by subtracting original ATP from total; compute ATP/ADP ratio |
| Components / Formulation | ATP Detection Reagent (recombinant luciferase, D-luciferin, Mg2+, cell lysis buffer, ATP stabilizers), ADP Converting Reagent (pyruvate kinase, phosphoenolpyruvate, conversion buffer), ATP standard (for calibration curve), detailed protocol |
| Storage Conditions | Lyophilized detection components at -20°C protected from light; reconstituted reagents at -20°C protected from light for 4 weeks; ATP standard at -20°C |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 100 assays, 500 assays, 1,000 assays (96-well format) |
| Product Form | Lyophilized and concentrated liquid reagent kit |
| Quality Control | Each lot standardized with known ATP and ADP reference solutions; linearity confirmed for ATP (R2 ≥0.99) and total adenine nucleotide measurements; ATP recovery >90% in spiked cell lysate samples |
| Key Features | Sequential single-well measurement of both ATP and ADP from identical sample; includes cell lysis for complete adenine nucleotide extraction; rapid protocol (10 minutes); luminescence signal stable for batch processing; valuable for assessing cellular bioenergetics beyond viability alone |
For research use only, not for clinical use.
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