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| Product Name | Alkaline Phosphatase (ALP) Activity Assay Kit, pNPP Substrate, Colorimetric |
| Catalog No. | ETR-HMM-0089 |
| Description | Colorimetric alkaline phosphatase (ALP) activity assay kit utilizing para-nitrophenyl phosphate (pNPP) as a chromogenic substrate. Upon dephosphorylation by ALP, pNPP is converted to para-nitrophenol (pNP), which ionizes to the yellow p-nitrophenolate anion under alkaline conditions with a strong absorbance at 405 nm. The kit is suitable for measuring ALP activity in serum, plasma, cell and tissue extracts, purified enzyme preparations, and for evaluating ALP expression in reporter gene assays (secreted alkaline phosphatase, SEAP). The optimized pNPP formulation ensures low spontaneous hydrolysis (blank stability) while providing excellent sensitivity for ALP detection. Includes an alkaline buffer with Mg2+ and Zn2+ cofactors, pNPP substrate tablets, stop solution, and pNP standard for absolute quantification. |
| Intended Use | Quantitative measurement of alkaline phosphatase activity in: clinical biochemistry (serum/plasma ALP for liver and bone function assessment in research); cell and tissue lysates; reporter gene expression (SEAP); osteoblast differentiation studies; quality control of recombinant ALP (calf intestinal, shrimp, bacterial ALP); ALP conjugation efficiency in ELISA and western blot; enzyme inhibitor screening. |
| Principle / Technology | p-Nitrophenyl phosphate (pNPP) is hydrolyzed by alkaline phosphatase to inorganic phosphate and p-nitrophenol (pNP). At the alkaline pH of the assay (pH 10.3-10.4), pNP (pKa 7.15) is predominantly in the yellow p-nitrophenolate anion form, which absorbs at 405 nm with a molar extinction coefficient of 18,500 M^-1 cm^-1. The rate of increase in absorbance at 405 nm is directly proportional to ALP activity. The reaction is stopped with NaOH, which further raises pH, fully ionizing pNP and inactivating the enzyme. |
| Detection Method | Add sample (serum, lysate, or purified enzyme) to assay buffer containing pNPP (1-5 mg/mL); incubate at 25-37 C, monitor A405 kinetically (every 30-60 s for 5-30 min) or as endpoint assay (15-60 min); stop with NaOH if endpoint; calculate activity using: U/mL = (delta A405/min x reaction volume x dilution factor) / (18.5 x sample volume), where 18.5 is the millimolar extinction coefficient of pNP at 405 nm. |
| Sample Type | Serum, plasma (heparin only — EDTA and citrate chelate Zn2+ and Mg2+ cofactors), cell lysates, tissue homogenates, purified alkaline phosphatase (CIAP, SAP, bacterial ALP), cell culture supernatant (SEAP), milk and dairy products (for pasteurization verification). |
| Performance Range / Specifications | Detection range: 0.001-1.0 U/mL ALP in cuvette (1 U = 1 umol pNPP hydrolyzed per minute at 37 C); linear range: up to 0.8 A405 (absorbance endpoint) or up to 0.05 delta A/min (kinetic mode); assay time: 5-30 min kinetic or 15-60 min endpoint at 37 C. |
| Sensitivity / LOD | Detection limit: 0.001 U/mL ALP (approximately 0.001 umol pNP/min/mL); corresponds to approximately 1 ng/mL purified calf intestinal ALP. |
| Specificity | pNPP is specific for alkaline phosphatase (optimum pH 10.3); acid phosphatases have negligible activity at this alkaline pH; protein tyrosine phosphatases and serine/threonine phosphatases show no activity with pNPP at pH 10.3. pNPP is also a substrate for some phosphodiesterases at neutral pH, but these are inactive at alkaline pH. |
| Reaction Conditions / Protocol | Standard reaction: pH 10.3-10.4, Mg2+ (1-5 mM) and Zn2+ (0.1-1 mM) as cofactors; incubation temperature 25-37 C; kinetic measurement at 405 nm for 5-30 min; endpoint: stop with 2 M NaOH (0.5-1.0 M final). |
| Components / Formulation | pNPP Substrate Tablets (5 mg/tablet, 20 tablets), Assay Buffer (5x, pH 10.3, 50 mL: diethanolamine buffer with MgCl2 and ZnCl2), Stop Solution (2 M NaOH, 25 mL), pNP Standard (1 mM, 1 mL — for standard curve), 96-well Clear Flat-Bottom Plate, Protocol. |
| Storage Conditions | pNPP tablets at -20 C desiccated (protect from light and moisture); Assay Buffer at 2-8 C; Stop Solution at RT; pNP Standard at 2-8 C protected from light. |
| Shelf Life | 12 months from date of manufacture; reconstituted pNPP substrate use within 1 day at 2-8 C (protect from light). |
| Package Specifications | 1 kit (sufficient for approximately 500 tests in 200 uL assay volume, 96-well plate format). |
| Product Form | Effervescent pNPP substrate tablets; liquid buffers; pNP standard solution. |
| Quality Control | Each lot tested with: calf intestinal ALP standard (linearity R2 >0.99 over 0-1 U/mL); pNPP substrate spontaneous hydrolysis rate <0.001 A405/h at 37 C; sensitivity: 0.001 U/mL detectable; lot-to-lot CV of ALP measurement <5%. |
| Key Features | pNPP tablet format for convenience and stability; kinetic and endpoint compatible; diethanolamine buffer for maximum activity; includes pNP standard for calibration; suitable for clinical research and biotechnology applications; 96-well plate compatible. |
| Purity | pNPP purity >=99% by HPLC; free pNP in substrate <0.1%; diethanolamine buffer low in trace metals (Fe <0.5 ppm, Cu <0.1 ppm). |
| Concentration | pNPP working concentration: 1-5 mg/mL (2.7-13.5 mM); pNP standard: 1 mM; Km of calf intestinal ALP for pNPP approximately 0.2-0.5 mM at pH 10.3. |
| Activity / Unit Definition | 1 ALP unit = 1 umol pNPP hydrolyzed per minute at 37 C in diethanolamine buffer pH 10.3; specific activity of calf intestinal ALP approximately 2,000-4,000 U/mg. |
| Molecular Weight | pNPP disodium salt hexahydrate: 371.14 g/mol; pNP: 139.11 g/mol; p-nitrophenolate: 139.11 g/mol. |
| Source / Origin | pNPP synthetic; all buffer components analytical grade; no animal-derived components except optional ALP enzyme provided for control purposes. |
| pH Range / Optimal pH | Optimal pH 10.3-10.4 (diethanolamine-HCl buffer); active range pH 9.0-11.0; pNP detection at alkaline pH (pKa 7.15, fully ionized above pH 9). |
| Shipping Conditions | Ambient temperature for buffers; pNPP tablets on cold pack. |
| Expiration Date / Stability | 12 months at recommended conditions; pNPP tablets stable at -20 C for 24 months; reconstituted substrate use same day. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. |
| Compatibility | Heparinized plasma is compatible; EDTA and citrate plasma interfere (chelate Zn2+ and Mg2+). Hemolyzed samples cause falsely elevated ALP due to erythrocyte ALP release. Inorganic phosphate (>10 mM) inhibits ALP. Chelating agents (EDTA, EGTA) must be avoided. Reducing agents (DTT, beta-mercaptoethanol up to 5 mM) do not interfere. Compatible with commercial lysis buffers (RIPA, NP-40, Triton X-100). For serum samples, physiological ALP activity ranges widely (30-120 U/L) and reference ranges vary by age, sex, and methodology. |
| Recommended Buffer System | 5x Assay Buffer: 1.0 M diethanolamine-HCl pH 10.3, 2.5 mM MgCl2, 0.5 mM ZnCl2; Stop Solution: 2 M NaOH; storage of substrate: 300 mM Tris-HCl pH 8.0, 1 mM MgCl2, 0.1 mM ZnCl2. |
| Application Notes / Precautions | Dissolve one pNPP tablet per 5 mL water to obtain 1 mg/mL substrate solution (or per 1-2 mL for 3-5 mg/mL). Protect pNPP solutions from light; store on ice and use within 8 hours. For kinetic assays, pre-warm assay buffer and plate to reaction temperature before adding sample. ALP from different sources has different optimal conditions: calf intestinal ALP (highest activity, 2,000-4,000 U/mg), bacterial ALP (lower activity, 50-100 U/mg), shrimp ALP (heat-labile, 500-1,000 U/mg). Include a substrate blank (buffer only, no enzyme) to correct for pNPP auto-hydrolysis. For serum samples, dilute 1:5 to 1:20 in assay buffer if activity exceeds the linear range. |
| Batch-to-Batch Consistency | ALP linearity R2 >0.99; pNPP auto-hydrolysis <0.001 A405/h; sensitivity 0.001 U/mL; lot-to-lot CV <5%. |
For research use only, not for clinical use.
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