QuantiChrom™ Alkaline Phosphatase Assay Kit

Protocol SDS

Application

• For quantitative determination of alkaline phosphatase (ALP) activity using stable p-nitrophenol phosphate substrate.

Key Features

• High sensitivity and wide linear range. Use 5 µL serum or plasma sample. The detection limit is 2 U/L, linear up to 800 U/L.
• Homogeneous and simple procedure. A simple “mix-and-measure” procedure allows reliable quantitation of ALP activity within 5 minutes.
• Robust and amenable to HTS. All reagents are compatible with high-throughput liquid handling instruments.

Method

• OD405nm

Samples

• Serum, plasma, etc

Species

• All

Procedure

• 4 min

Size

• 250 tests

Detection Limit

• 2 U/L

Shelf Life

• 12 months

More Details

Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate esters in an alkaline environment, resulting in the formation of an organic radical and inorganic phosphate. In mammals, this enzyme is found mainly in the liver and bones. A marked increase in serum ALP levels, a disease known as hyperalkalinephosphatasemia, has been associated with malignant biliary obstruction, primary biliary cirrhosis, primary sclerosing cholangitis, hepatic lymphoma, and sarcoidosis. Simple, direct, and automation-ready procedures for measuring ALP activity in serum are becoming popular in Research and Drug Discovery. BioAssay Systems QuantiChrom™ Alkaline Phosphatase Assay Kit is designed to measure ALP activity directly in biological samples without pretreatment. The improved method utilizes p-nitrophenyl phosphate that is hydrolyzed by ALP into a yellow-colored product (maximal absorbance at 405nm). The rate of the reaction is directly proportional to the enzyme activity.

Can the calibrator and water blank be measured after the samples, and if so how long after?

We recommend measuring calibrator and water blank simultaneously with the samples, to avoid any potential sources of error, but it is not essential. Potential sources of error that can be introduced by non-simultaneous measurement are: variations in pipetting volumes, variation in microplate reader performance, evaporation of reagents caused by long incubation, etc.

There are yellow chrystals in the pNPP vial – can I still use the product?

Yes, but you first have to dissolve the crystals. The yellow crystals are phenol which has separated from the reagent during the freezing process. Heat the vial until all phenol crystals disappear (melting point is around 40 °C), and mix well before use.

Is pNPP light sensitive? Does it need to be handled in the dark room only, and dark colored 96well plates necessary?

pNPP is light sensitive and must be stored protected from light, but it can be used under normal laboratory conditions. Only clear flat bottom microplates should be used. Do not use plates, where the bottom is not clear and flat.

How do I measure ALP activity animal tissue?

Homogenize tissue sample in 10 mM Tris Buffer (pH 8.5), 50 mM NaCl, or lysis buffer of your choice.

– The assay is compatible with up to 2% SDS or Tween-20 in the sample.

– The assay is incompatible with EDTA, oxalate, fluoride, citrate.

– Centrifuge homogenized tissue sample at 10,000 x g for 10 min at 4°C.

– Use the clear supernatant for alkaline phosphatase assay.

Sul, et al. (2020). Atherogenic diet-induced bone loss is primarily due to increased osteoclastogenesis in mice. The Journal of Nutritional Biochemistry. 79: 108337. Assay: Alkaline Phosphatase in mouse cells.

Harb, S. V., et al. (2020). Hydroxyapatite and β-TCP modified PMMA-TiO2 and PMMA-ZrO2 coatings for bioactive corrosion protection of Ti6Al4V implants. Materials Science and Engineering: C. 116: 111149. Assay: Alkaline Phosphatase in human osteoblast.

Harper, E., et al. (2020). RANKL treatment of vascular endothelial cells leading to paracrine pro-calcific signaling involves ROS production. Molecular and Cellular Biochemistry. 464(1-2): 111-117. Assay: Alkaline Phosphatase in human cell lysate.

Jeffrey, J. D., et al. (2019). Physiological status of silver carp (Hypophthalmichthys molitrix) in the Illinois River: An assessment of fish at the leading edge of the invasion front. Comparative Biochemistry and Physiology. Part D, Genomics & Proteomics. 32, 100614. Assay: Alkaline Phosphatase in silver carp plasma.

Martineau, C., et al. (2020). Preclinical safety and efficacy of 24R,25-dihydroxyvitamin D3 or lactosylceramide treatment to enhance fracture repair. Journal of Orthopaedic Translation. 23: 77-88. Assay: Alkaline Phosphatase in mouse serum.

Zheng, Z., et al. (2020). Magnesium-organic framework-based stimuli-responsive systems that optimize the bone microenvironment for enhanced bone regeneration. Chemical Engineering Journal. 396: 125241. Assay: Alkaline Phosphatase in rat bone marrow mesenchymal stem cells.

Moghazy, H., et al. (2020). Protective Effect of Oxytocin Against Bone Loss in a Female Rat Model of Osteoporosis. Reports of Biochemistry & Molecular Biology. 9(2): 147-155. Assay: Alkaline Phosphatase in rat serum.

Pasqualini, D., et al. (2020). Osteogenic potential of fast set bioceramic cements: Molecular and in vitro study. Applied Sciences. 10(19): 6713. Assay: Alkaline Phosphatase in human fibroblast.

Niedermair, T., et al. (2020). Absence of α-calcitonin gene-related peptide modulates bone remodeling properties of murine osteoblasts and osteoclasts in an age-dependent way. Mechanisms of Ageing and Development, 189, 111265. Assay: Alkaline Phosphatase in mouse osteoblast.

Kim, H.-W., Kim, Y.-J. (2021). Fabrication of strontium-substituted hydroxyapatite scaffolds using 3D printing for enhanced bone regeneration. Journal of Materials Science. 56(2): 1673-1684. Assay: Alkaline Phosphatase in mouse osteoblast.

Liu, J., et al. (2020). Stem cells in the periodontal ligament differentiated into osteogenic, fibrogenic and cementogenic lineages for the regeneration of the periodontal complex. Journal of Dentistry. 92: 103259. Assay: Alkaline Phosphatase in human periodontal ligament stem cell lysate.

Xu, L., et al. (2020). Hypoxia influences the effects of magnesium degradation products on the interactions between endothelial and mesenchymal stem cells. Acta Biomaterialia. 101: 624-636. Assay: Alkaline Phosphatase in human umbilical cord/vein cells.

Wu, Q., et al. (2020). The sialylation profile of IgG determines the efficiency of antibody directed osteogenic differentiation of iMSCs by modulating local immune responses and osteoclastogenesis. Acta Biomaterialia. 114: 221-232. Assay: Alkaline Phosphatase in rat induced pluripotent stem cell derived mesenchymal stem cell.

Wang, Q., et al. (2020). Effects of degradable magnesium on paracrine signaling between human umbilical cord perivascular cells and peripheral blood mononuclear cells. Biomaterials Science. 8(21): 5969-5983. Assay: Alkaline Phosphatase in human umbilical cord perivascular cells .

Wang, Q., et al. (2020). Macrophage-derived oncostatin M/bone morphogenetic protein 6 in response to Mg-based materials influences pro-osteogenic activity of human umbilical cord perivascular cells. Acta Biomaterialia. Assay: Alkaline Phosphatase in human umbilical cord perivascular cells .

Zhao, Z., et al. (2019). Human periodontal ligament stem cell seeding on calcium phosphate cement scaffold delivering metformin for bone tissue engineering. Journal of Dentistry. 91: 103220. Assay: Alkaline Phosphatase in human cell lysate.

Zhao, Z., et al (2019). Human periodontal ligament stem cells on calcium phosphate scaffold delivering platelet lysate to enhance bone regeneration. RSC Advances, 9(70), 41161-41172. Assay: Alkaline Phosphatase in human cell lysate.

Liu, X., et al. (2020). Injectable electrical conductive and phosphate releasing gel with two-dimensional black phosphorus and carbon nanotubes for bone tissue engineering. ACS Biomaterials Science & Engineering. 6(8): 4653-4665. Assay: Alkaline Phosphatase in human cell lysate.

Hann, S. Y., et al. (2021). Dual 3D printing for vascularized bone tissue regeneration. Acta Biomaterialia. Assay: Alkaline Phosphatase in human mesenchymal stem cells.

Chen, H., et al. (2020). An antibacterial and injectable calcium phosphate scaffold delivering human periodontal ligament stem cells for bone tissue engineering. RSC Advances. 10(66): 40157-40170. Assay: Alkaline Phosphatase in human cell lysate.

Niedermair, T., et al. (2020). Impact of the sensory and sympathetic nervous system on fracture healing in ovariectomized mice. International Journal of Molecular Sciences. 21(2): 405. Assay: Alkaline Phosphatase in mouse serum.

Murali, V. P., et al. (2020). Modified electrospun chitosan membranes for controlled release of simvastatin. International Journal of Pharmaceutics. 584: 119438. Assay: Alkaline Phosphatase in mouse cell lysate.

Niedermair, T., et al. (2020). Influence of extracellular vesicles isolated from osteoblasts of patients with cox-arthrosis and/or osteoporosis on metabolism and osteogenic differentiation of bmscs. Frontiers in Bioengineering and Biotechnology. 8: 615520. Assay: Alkaline Phosphatase in human osteoblast.

George, M. N., et al. (2020). Phosphate functionalization and enzymatic calcium mineralization synergistically enhance oligo[poly(Ethylene glycol) fumarate] hydrogel osteoconductivity for bone tissue engineering. Journal of Biomedical Materials Research Part A, 108(3), 515-527. Assay: Alkaline Phosphatase in mouse preosteoblast.

Ji, S., Guvendiren, M. (2019). 3D printed wavy scaffolds enhance mesenchymal stem cell osteogenesis. Micromachines. 11(1): 31. Assay: Alkaline Phosphatase in human mesenchymal stem cell lysate.

Liu, X., et al. (2020). 3D-printed scaffolds with carbon nanotubes for bone tissue engineering: Fast and homogeneous one-step functionalization. Acta Biomaterialia. 111: 129-140. Assay: Alkaline Phosphatase in mouse cell lysate.

Liang, Z. C., et al. (2020). Carboxylic acid-functionalized polycarbonates as bone cement additives for enhanced and sustained release of antibiotics. Journal of Controlled Release. Assay: Alkaline Phosphatase in mouse cells.

Fasina, YO, Thanissery, RR (2011). Comparative efficacy of a yeast product and bacitracin methylene disalicylate in enhancing early growth and intestinal maturation in broiler chicks from breeder hens of different ages. Poult Sci. 90(5):1067-73. Assay: Alkaline phosphatase in chicken Small intestine tissue.

Gaharwar AK, et al (2011). Assessment of using laponite cross-linked poly(ethylene oxide) for controlled cell adhesion and mineralization. Acta Biomater. 7(2):568-77. Assay: Alkaline phosphatase in mouse preosteoblast cells.

Hutchins HL, et al (2011). Eicosapentaenoic acid decreases expression of anandamide synthesis enzyme and cannabinoid receptor 2 in osteoblast-like cells. J Nutr Biochem. 22(2):195-200. Assay: Alkaline phosphatase in human osteoblast.

Katuru R, et al (2011). Mevalonate depletion mediates the suppressive impact of geranylgeraniol on murine B16 melanoma cells. Exp Biol Med (Maywood) 236(5):604-13. Assay: Alkaline phosphatase in mouse cell lysate.

Kucukalic-Selimovic E, et al (2011). Evaluation of bone remodelling parameters after one year treatment with alendronate in postmenopausal women with osteoporosis. Bosn J Basic Med Sci. 11(1):41-5. Assay: Alkaline phosphatase in human serum.

Palic, D et al (2011). Use of rapid cytochemical staining to characterize fish blood granulocytes in species of special concern and determine potential for function testing. Fish Shellfish Immunol. 30(2):646-52. Assay: ALP in fish Granulocytes.

Sekiya, S, Suzuki, A (2011). Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature 475(7356):390-3. Assay: Alkaline phosphatase in mouse plasma.

Wang Y, et al (2011). Small interfering RNA knocks down the molecular target of alendronate, farnesylpyrophosphate synthase, in osteoclast and osteoblast cultures. Mol Pharm. 8(4):1016-24. Assay: Alkaline phosphatase in murine bone marrow.

Zhi L, et al (2011). Synergistic effect of recombinant human bone morphogenic protein-7 and osteogenic differentiation medium on human bone-marrow-derived mesenchymal stem cells in vitro. Int Orthop. 35(12), 1889-1895. Assay: Alkaline phosphatase in stem cells culture.

Kauther MD, et al (2010). Alpha-calcitonin gene-related peptide can reverse the catabolic influence of UHMWPE particles on RANKL expression in primary human osteoblasts. Int J Biol Sci.6(6):525-36. Assay: Alkaline phosphatase in human osteoblasts.

Oldinski, RA et al (2010). Synthesis and characterization of a Hyaluronan-polyethylene copolymer for biomedical applications. J Biomed Mater Res B Appl Biomater. 94(2):441-6. Assay: Alkaline phosphatase in human cells.

Ponnapakkam T, et al (2010). A treatment trial of vitamin D supplementation in breast-fed infants: universal supplementation is not necessary for rickets prevention in Southern Louisiana. Clin Pediatr (Phila). 49(11):1053-60. Assay: Alkaline phosphatase in human blood.

Keskar V, et al (2009). In vitro evaluation of macroporous hydrogels to facilitate stem cell infiltration, growth, and mineralization. Tissue Eng Part A. 15(7):1695-707. Assay: Alkaline phosphatase in human hydrogels.

Maskiewicz, R (2009). SUBLIMABLE SUSTAINED RELEASE DELIVERY SYSTEM AND METHOD OF MAKING SAME. US Patent application number: 20090220602. Assay: Alkaline phosphatase in enzyme alkaline phosphatase.

Milne TJ, et al (2009). Induction of osteopenia during experimental tooth movement in the rat: alveolar bone remodelling and the mechanostat theory. Eur J Orthod. 31(3):221-31. Assay: Alkaline phosphatase in rat serum.

Panizo S,et al (2009). RANKL increases vascular smooth muscle cell calcification through a RANK-BMP4-dependent pathway. Circ Res. 104(9):1041-8. Assay: Alkaline phosphatase in rat cells.

Richard Maskiewicz (2009). Sublimable Sustained Release Delivery System And Method Of Making Sameus Patent Application Number: 20090220602. Assay: Alkaline phosphatase in enzyme alkaline phosphatase.

Henderson JA, et al (2008). Concurrent differentiation of marrow stromal cells to osteogenic and vasculogenic lineages. Macromol Biosci. 8(6):499-507. Assay: Alkaline phosphatase in rat bone marrow.

Bhattacharya, A. et al (2007). Effect of fish oil on bone mineral density in aging C57BL/6 female mice. J. Nutr. Biochem 18(6):372-379. Assay: Alkaline phosphatase in mouse serum.

Wan, Y. et al (2007). PPAR-g regulates osteoclastogenesis in mice. Nature Med. 13(12): 1496-1503. Assay: Alkaline phosphatase in mouse serum.

Bhattacharya, A et al (2006). Effect of fish oil on bone mineral density in aging C57BL/6 female mice. J. Nutr. Biochem 18(6):372-379. Assay: ALP in mouse serum.

Kim HJ, et al (2006). Glucocorticoids suppress bone formation via the osteoclast. J Clin Invest. 116(8):2152-60. Assay: Alkaline phosphatase in mouse serum.

Lian Q, et al (2006). Establishing clonal cell lines with endothelial-like potential from CD9(hi), SSEA-1(-) cells in embryonic stem cell-derived embryoid bodies. PLoS One 1:e6. Assay: Alkaline phosphatase in mouse Stem cell line.

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If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.

– Fast turnaround
– Quality data
– Low cost

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