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| Product Name | Alkaline Phosphatase (ALP) Activity Colorimetric Assay Kit |
| Catalog No. | ETR-HMM-0057 |
| Description | A colorimetric assay kit for measuring alkaline phosphatase enzymatic activity in biological samples using the chromogenic substrate para-nitrophenyl phosphate (pNPP). ALP hydrolyzes pNPP at alkaline pH to produce para-nitrophenol (pNP), a yellow product whose absorbance is measured at 405 nm. |
| Intended Use | Quantitative measurement of ALP activity in serum, plasma, cell lysates, and tissue homogenates for clinical chemistry biomarker research, osteoblast differentiation monitoring, liver and bone disease studies, and enzyme immunoassay normalization. |
| Principle / Technology | Alkaline phosphatase is a ubiquitous metalloenzyme that catalyzes the hydrolysis of phosphate monoesters at alkaline pH (optimal pH 9.5–10.5); the synthetic substrate pNPP (para-nitrophenyl phosphate disodium salt) is cleaved by ALP at the phosphate ester bond to release inorganic phosphate and para-nitrophenol; pNP is yellow in alkaline solution (molar absorption coefficient approximately 18,000 M-1cm-1 at 405 nm). |
| Detection Method | Colorimetric kinetic or endpoint absorbance measurement at 405 nm using a microplate spectrophotometer |
| Sample Type | Serum and plasma (human, rodent, and other mammalian species), cell lysates (osteoblasts, hepatocytes, intestinal epithelial cells), tissue homogenates (bone, liver, kidney, intestine), conditioned cell culture medium |
| Performance Range / Specifications | pNP standard curve: 0.01–10 μmol/mL; ALP activity range: 1–1,000 U/L depending on dilution; typical kinetic rate 0.001–1 OD/min at 37°C; one unit of ALP activity is defined as the amount producing 1 μmol pNP per minute at 37°C, pH 9.8 |
| Sensitivity / LOD | Lower detection limit: approximately 0.1 U/L in serum with 100 μL sample input; sensitivity equivalent to approximately 0.5 pmol pNP per minute per well in the microplate format |
| Specificity | pNPP is hydrolyzed specifically by phosphate monoesterases (ALP, acid phosphatase); ALP activity is maximally expressed at alkaline pH (pH 9.8 in this assay) where acid phosphatase is inactive; zinc and magnesium ions in the buffer cofactor mix are specifically required for ALP activity and are absent from the substrate-only blank |
| Reaction Conditions / Protocol | Dilute samples in provided Sample Dilution Buffer; add 80 μL Assay Buffer to microplate wells; add 10 μL diluted sample or pNP standards; start reaction by adding 10 μL pNPP Substrate Solution; incubate at 37°C for 15–30 minutes (kinetic) or exactly 60 minutes (endpoint); for endpoint: add 20 μL Stop Solution (0.5 M NaOH); measure absorbance at 405 nm; calculate activity from pNP standard curve or using ε = 18,000 M-1cm-1 |
| Components / Formulation | Assay Buffer (DEA buffer pH 9.8 with MgCl2 and ZnCl2), pNPP Substrate Solution (20 mM), pNP Standard (1 μmol/mL), Sample Dilution Buffer, Stop Solution (0.5 M NaOH), positive control (purified bovine intestinal ALP) |
| Storage Conditions | Assay Buffer and Substrate at 2–8°C protected from light; pNP Standard at -20°C for 12 months; all components stable for 12 months at specified conditions |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 200 assays, 500 assays, 1,000 assays (96-well format) |
| Product Form | Liquid ready-to-use assay buffer and substrate solutions with lyophilized standards |
| Quality Control | Each lot tested against purified bovine intestinal ALP reference material; specific activity within defined range; inter-assay CV <8% for serum reference samples; pNP standard curve R2 ≥0.999 |
| Key Features | Single-substrate assay requiring no radioactive materials; both kinetic and endpoint measurement modes; applicable to a broad range of mammalian ALP sources including serum, osteoblast cultures, and intestinal tissue; compatible with ALP isozyme differentiation using L-phenylalanine inhibitor |
For research use only, not for clinical use.
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