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| Product Name | ATPase Activity Assay Kit, Colorimetric, Malachite Green, High Sensitivity |
| Catalog No. | ETR-HMM-0087 |
| Description | High-sensitivity colorimetric assay kit for measuring ATPase (adenosine triphosphatase) activity in purified enzyme preparations, cell and tissue extracts, and membrane fractions. The assay is based on the detection of inorganic phosphate (Pi) released from ATP hydrolysis using the malachite green-molybdate method, which forms a stable green chromophore (absorbance at 620-650 nm) proportional to the amount of free phosphate. Unlike NADH-coupled spectrophotometric methods, this assay directly measures Pi and is compatible with any ATPase type including Na+/K+-ATPase, Ca2+-ATPase, H+-ATPase, myosin ATPase, and other ATP-hydrolyzing enzymes without interference from the intrinsic absorbance of nucleotide cofactors. The high-sensitivity formulation detects as little as 2 pmol of phosphate in a 100 uL reaction volume. |
| Intended Use | Quantification of ATPase activity in purified proteins (myosin, kinesin, dynein, ABC transporters), membrane preparations (plasma membrane, sarcoplasmic reticulum, mitochondrial inner membrane), tissue homogenates, and cell lysates for enzyme kinetics, inhibitor screening, drug discovery, and mechanistic studies of energy-transducing enzymes. |
| Principle / Technology | ATP hydrolysis by ATPase: ATP + H2O -> ADP + Pi. Released inorganic phosphate reacts with ammonium molybdate in acidic solution to form phosphomolybdic acid, which is then reduced by malachite green to form a green chromophore (phosphomolybdate-malachite green complex) with maximum absorbance at 620-650 nm. The color intensity is directly proportional to Pi concentration. An acid precipitation step stops the enzymatic reaction and simultaneously releases protein-bound phosphate. |
| Detection Method | Incubate enzyme sample with ATP substrate in assay buffer (37 C, 10-60 min); stop reaction by adding Malachite Green Reagent A (acid-molybdate); incubate 10 min at RT for color development (add stabilizer if needed); measure absorbance at 620-650 nm; calculate Pi concentration from standard curve (0-40 uM Pi); calculate ATPase activity as nmol Pi released/min/mg protein or per unit volume. |
| Sample Type | Purified ATPase enzymes, tissue homogenates (brain, kidney, heart, liver, skeletal muscle), cell lysates, membrane fractions (microsomes, plasma membrane preparations), mitochondrial fractions, myofibrillar preparations. |
| Performance Range / Specifications | Detection range: 2-2,000 pmol Pi per well (0.02-20 uM Pi in 100 uL); linear range: 0.02-10 uM Pi (R2 >0.99); typical assay time: 10-60 min enzymatic reaction + 10-20 min detection; compatible protein range: 0.1-50 ug protein per well. |
| Sensitivity / LOD | Detection limit: 2 pmol Pi (equivalent to 0.02 uM in 100 uL), approximately 5-10x more sensitive than standard malachite green assays; for ATPase activity: approximately 0.05 nmol Pi/min/mg protein detectable. |
| Specificity | Malachite green-molybdate specifically detects inorganic orthophosphate (Pi); does not detect ATP, ADP, AMP, or other organic phosphates (phosphocreatine, phosphoenolpyruvate, glucose-6-phosphate) at standard concentrations (<1 mM); moderate interference from labile organic phosphates (creatine phosphate at >5 mM) due to spontaneous hydrolysis in acid conditions. |
| Reaction Conditions / Protocol | Enzymatic reaction: 10-60 min at desired temperature (25-37 C) in 50 uL volume; color development: 10-20 min at RT; total assay time: 30-90 min. |
| Components / Formulation | ATP Substrate (100 mM in water, 0.5 mL), Phosphate Standard (10 mM KH2PO4, 0.5 mL), Malachite Green Reagent A (ammonium molybdate in sulfuric acid, 10 mL), Malachite Green Reagent B (malachite green oxalate, 0.2 mL — mix with Reagent A before use), Assay Buffer (5x, 25 mL), 96-well Clear Bottom Microplate, Protocol. |
| Storage Conditions | ATP substrate at -20 C (aliquot to avoid freeze-thaw); all other components at 4 C (2-8 C); protect malachite green reagents from light; mixed color reagent stable for 2-3 hours at RT. |
| Shelf Life | 12 months from date of manufacture; reconstituted ATP stable at -20 C for 6 months; mixed color reagent use within 3 hours. |
| Package Specifications | 1 kit (sufficient for 500 tests in 96-well plate format, 100 uL assay volume). |
| Product Form | Liquid reagents; ATP supplied as frozen liquid; phosphate standard as frozen liquid. |
| Quality Control | Each lot tested with: Na+/K+-ATPase standard from porcine cerebral cortex (specific activity 0.5-2 umol Pi/min/mg); linearity of Pi standard curve (R2 >0.99 over 0-20 uM); sensitivity: A620 of 2 uM Pi >=0.1; low background: A620 of blank <0.1; inter-assay CV <8%. |
| Key Features | Malachite green colorimetric detection; 2 pmol phosphate sensitivity; measures Pi directly (no coupled enzymes); compatible with all ATPase types; stable color; 96-well plate format for HTS. |
| Purity | All reagents: analytical grade or higher; water used: ultrapure (18.2 MOhm-cm); phosphate standard traceable to NIST SRM. |
| Concentration | ATP substrate: 100 mM; phosphate standard: 10 mM (10,000 pmol/uL); working range 0.02-20 uM Pi in assay. |
| Activity / Unit Definition | Detection of ATPase activity from 0.05-50 nmol Pi/min per well; measurable activity in as little as 0.1 ug membrane protein for high-activity preparations. |
| Molecular Weight | ATP disodium salt: 551.14 g/mol; KH2PO4: 136.09 g/mol; malachite green oxalate: 463.50 g/mol. |
| Source / Origin | Synthetic and fermentation-derived chemicals; ATP from microbial fermentation; phosphate standard traceable to NIST; all reagents animal-free. |
| pH Range / Optimal pH | Assay buffer pH 7.4 (physiological); color development in acidic conditions (HCl/H2SO4, pH ~1); optimal Pi detection pH <1. |
| Shipping Conditions | Cold pack (2-8 C) for malachite green reagents; ATP and phosphate standard on dry ice (-20 C). |
| Expiration Date / Stability | 12 months at recommended conditions; thawed ATP and phosphate standard can be stored at 2-8 C for 2 weeks or refrozen at -20 C. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. |
| Compatibility | Compatible with most common buffer systems (Tris, HEPES, PBS, MOPS, imidazole) provided they are phosphate-free. Any phosphate-containing buffer (PBS, phosphate buffer) will produce high background and must be avoided or exchanged by dialysis/desalting before assay. Reducing agents (DTT, beta-mercaptoethanol >1 mM) interfere with color development (reduce phosphomolybdate). Detergents: Triton X-100, NP-40, SDS >0.1% may interfere; Tween-20 up to 0.05% is tolerated. Compatible with 96-well and 384-well clear-bottom plates. |
| Recommended Buffer System | 5x Assay Buffer: 250 mM Tris-HCl pH 7.4, 500 mM NaCl, 25 mM KCl, 25 mM MgCl2; color reagent: 0.045% malachite green, 4.2% ammonium molybdate in 4 M HCl. |
| Application Notes / Precautions | All glassware and plasticware must be phosphate-free; wash with phosphate-free detergent and rinse thoroughly with ultrapure water. Use disposable plastic cuvettes or 96-well plates. Include ouabain (1 mM) or orthovanadate (1 mM) to verify specific P-type ATPase activity. For membrane preparations, determine specific activity as ouabain-sensitive (Na+/K+-ATPase), thapsigargin-sensitive (SERCA Ca2+-ATPase), or bafilomycin-sensitive (V-ATPase) Pi release. Include enzyme blank (no ATP) and substrate blank (no enzyme) controls. The color is stable for 1-2 hours at RT; measure absorbance within this window. Prepare fresh mixed color reagent daily. |
| Batch-to-Batch Consistency | Pi standard curve slope within +/-10% of reference lot; sensitivity (2 uM Pi absorbance) within +/-15%; blank absorbance <0.1. |
For research use only, not for clinical use.
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