Alkaline Phosphatase Assay Kit

Produktnummer ABIN1000247

Produktdetails

Target: Alkaline Phosphatase (ALP)

Detektionsbereich: 2-800 U/L

Untere Nachweisgrenze: 2 U/L

Applikation: Activity Assay (AcA)

Proben: Serum, Plasma

Produktmerkmale: 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.
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.

Bestandteile: Assay Buffer: 50 mL, pH 10.5. Mg Acetate: 1.5 mL. 0.2 M pNPP Liquid: 600 µL. 1 M Calibrator: 10 mL Tartrazine.

Benötigtes Material: Pipeting devices and accessories (e.g. multi-channel pipettor). Clear bottom 96-well plates (e.g. Corning Costar) and plate reader.

Anwendungsinformationen

Applikationshinweise: Direct Assays: ALP activity in serum, plasma and other sources.
Characterization and Quality Control for ALP production.
Drug Discovery: high-throughput screen for ALP inhibitors and evaluation of ALP inhibitors.

Protokoll: This assay is based on a kinetic reaction. Use of a multi-channel pipettor is recommended. Addition of Working Reagent to samples should be quick and mixing should be brief but thorough. Assays can be executed at room temperature or 37°C.

Procedure using 96-well plate:
1. Transfer 200 µL distilled water (H2O) and 200 µL Calibrator into separate wells of a clear bottom 96-well plate.
2. Carefully transfer 5 to 50 µL samples into other wells.
3. Pipet 150 to 195 µL Working Solution to sample wells. The final reaction volume in the sample wells should be 200 µL. Tap plate briefly to mix.
4. Read OD405nm (t = 0), and again after 4 min (t = 4 min) on a plate reader.

Procedure using Cuvette:
1. Transfer 50 µL samples into 1-cm cuvettes.
2. Pipet 950 µL Working Solution to samples. Mix briefly.
3. Read OD405nm shortly after the mixing, and again after 4 min.

Aufbereitung der Reagenzien:

Equilibrate reagents to room temperature. The Working Solution is prepared by mixing for each 96-well assay, 200 µL Assay Buffer, 5 µL Mg Acetate (final 5 mM) and 2 µL pNPP liquid substrate (10 mM). Fresh reconstitution is recommended, although the Working Solution is stable for at least one day at room temperature.

Aufbereitung der Proben:

ALP is stable for 48 hours at 4°C and 2 months at - 20°C. EDTA, oxalate, fluoride, citrate are known inhibitors of ALP and should be avoided in sample preparation. Serum, plasma (no EDTA/citrate, ideally unhemolyzed) and cell culture media can be assayed directly. To measure intracellular ALP, cell lysate can be prepared : 10 4 cells are washed with PBS and lysed in 0.5 mL 0.2% Triton X-100 in distilled water by shaking for 20 min at room temperature.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C

Referenzen

Gaharwar, Schexnailder, Kline, Schmidt: "Assessment of using laponite cross-linked poly(ethylene oxide) for controlled cell adhesion and mineralization." in: Acta biomaterialia, Vol. 7, Issue 2, pp. 568-77, (2011) (PubMed).

Hutchins, Li, Hannon, Watkins: "Eicosapentaenoic acid decreases expression of anandamide synthesis enzyme and cannabinoid receptor 2 in osteoblast-like cells." in: The Journal of nutritional biochemistry, Vol. 22, Issue 2, pp. 195-200, (2011) (PubMed).

Wang, Panasiuk, Grainger: "Small interfering RNA knocks down the molecular target of alendronate, farnesyl pyrophosphate synthase, in osteoclast and osteoblast cultures." in: Molecular pharmaceutics, Vol. 8, Issue 4, pp. 1016-24, (2011) (PubMed).

Palić, Beck, Palić, Andreasen: "Use of rapid cytochemical staining to characterize fish blood granulocytes in species of special concern and determine potential for function testing." in: Fish & shellfish immunology, Vol. 30, Issue 2, pp. 646-52, (2011) (PubMed).

Kučukalić-Selimović, Valjevac, Hadžović-Džuvo, Skopljak-Beganović, Alimanovic-Alagić, Brković: "Evaluation of bone remodelling parameters after one year treatment with alendronate in postmenopausal women with osteoporosis." in: Bosnian journal of basic medical sciences / Udruženje basičnih mediciniskih znanosti = Association of Basic Medical Sciences, Vol. 11, Issue 1, pp. 41-5, (2011) (PubMed).

Zhi, Chen, Pang, Uludag, Jiang: "Synergistic effect of recombinant human bone morphogenic protein-7 and osteogenic differentiation medium on human bone-marrow-derived mesenchymal stem cells in vitro." in: International orthopaedics, Vol. 35, Issue 12, pp. 1889-95, (2011) (PubMed).

Fasina, Thanissery: "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." in: Poultry science, Vol. 90, Issue 5, pp. 1067-73, (2011) (PubMed).

Katuru, Fernandes, Elfakhani, Dutta, Mills, Hynds, King, Mo: "Mevalonate depletion mediates the suppressive impact of geranylgeraniol on murine B16 melanoma cells." in: Experimental biology and medicine (Maywood, N.J.), Vol. 236, Issue 5, pp. 604-13, (2011) (PubMed).

Sekiya, Suzuki: "Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors." in: Nature, Vol. 475, Issue 7356, pp. 390-3, (2011) (PubMed).

Oldinski, Cranson, James: "Synthesis and characterization of a Hyaluronan-polyethylene copolymer for biomedical applications." in: Journal of biomedical materials research. Part B, Applied biomaterials, Vol. 94, Issue 2, pp. 441-6, (2010) (PubMed).

Ponnapakkam, Bradford, Gensure: "A treatment trial of vitamin D supplementation in breast-fed infants: universal supplementation is not necessary for rickets prevention in Southern Louisiana." in: Clinical pediatrics, Vol. 49, Issue 11, pp. 1053-60, (2010) (PubMed).

Kauther, Xu, Wedemeyer: "Alpha-calcitonin gene-related peptide can reverse the catabolic influence of UHMWPE particles on RANKL expression in primary human osteoblasts." in: International journal of biological sciences, Vol. 6, Issue 6, pp. 525-36, (2010) (PubMed).

Keskar, Marion, Mao, Gemeinhart: "In vitro evaluation of macroporous hydrogels to facilitate stem cell infiltration, growth, and mineralization." in: Tissue engineering. Part A, Vol. 15, Issue 7, pp. 1695-707, (2009) (PubMed).

Panizo, Cardus, Encinas, Parisi, Valcheva, López-Ongil, Coll, Fernandez, Valdivielso: "RANKL increases vascular smooth muscle cell calcification through a RANK-BMP4-dependent pathway." in: Circulation research, Vol. 104, Issue 9, pp. 1041-8, (2009) (PubMed).

Milne, Ichim, Patel, McNaughton, Meikle: "Induction of osteopenia during experimental tooth movement in the rat: alveolar bone remodelling and the mechanostat theory." in: European journal of orthodontics, Vol. 31, Issue 3, pp. 221-31, (2009) (PubMed).

Henderson, He, Jabbari: "Concurrent differentiation of marrow stromal cells to osteogenic and vasculogenic lineages." in: Macromolecular bioscience, Vol. 8, Issue 6, pp. 499-507, (2008) (PubMed).

Stewart, Byron, Pondenis, Stewart: "Effect of dexamethasone supplementation on chondrogenesis of equine mesenchymal stem cells." in: American journal of veterinary research, Vol. 69, Issue 8, pp. 1013-21, (2008) (PubMed).

Bhattacharya, Rahman, Sun, Fernandes: "Effect of fish oil on bone mineral density in aging C57BL/6 female mice." in: The Journal of nutritional biochemistry, Vol. 18, Issue 6, pp. 372-9, (2007) (PubMed).

Lian, Yeo, Que, Tan, Yu, Yin, Salto-Tellez, Menshawe El Oakley, Lim: "Establishing clonal cell lines with endothelial-like potential from CD9(hi), SSEA-1(-) cells in embryonic stem cell-derived embryoid bodies." in: PLoS ONE, Vol. 1, pp. e6, (2007) (PubMed).

Wan, Chong, Evans: "PPAR-gamma regulates osteoclastogenesis in mice." in: Nature medicine, Vol. 13, Issue 12, pp. 1496-503, (2007) (PubMed).

Antigendetails

Target: Alkaline Phosphatase (ALP)

Andere Bezeichnung: Alkaline Phosphatase (ALP Produkte)

Synonyme: ALP Kit, ALP1 Kit, CRASH Kit, ECK0378 Kit, JW0374 Kit, psiA Kit, 2410004D18Rik Kit, AU040643 Kit, AW060375 Kit, ILC Kit, CTACK Kit, ESkine Kit, Akp2 Kit, alp Kit, zgc:56672 Kit, DDBDRAFT_0205491 Kit, DDBDRAFT_0231570 Kit, DDB_0205491 Kit, DDB_0231570 Kit, Actn2lp Kit, Alp Kit, asparaginase like 1 Kit, bacterial alkaline phosphatase Kit, C-C motif chemokine ligand 27 Kit, alkaline phosphatase, liver/bone/kidney Kit, alkaline phosphatase Kit, PDZ and LIM domain 3 Kit, ASRGL1 Kit, phoA Kit, Asrgl1 Kit, CCL27 Kit, alpl Kit, alp Kit, Pdlim3 Kit

Hintergrund: Quantitative determination of alkaline phosphatase ALP activity using stable p-nitrophenol phosphate substrate at 405nm.

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. 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. The 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. p-Nitrophenyl phosphate p-nitrophenol + phosphate