p-Nitrophenyl Phosphate (pNPP, KR-pT-IRR), Phosphatase Assay Kit, BioAssay(TM)

Description:\nPara-nitrophenyl phosphate (pNPP) is a chromogenic substrate for most phosphatases such as alkaline phosphatases, acid phosphatases, protein tyrosine phosphatases and serine/threonine phosphatases. The reaction yields para-nitrophenol, which becomes an intense yellow soluble product under alkaline conditions and can be conveniently measured at 405 nm on a spectrophotometer. phosphatase p-Nitrophenyl phosphate p-nitrophenol + phosphate\nThis homogeneous "mix-and-measure" assay involves simply adding a single reagent to the phosphatase and measuring the product formation using any absorbance reader. The assay can be conveniently performed in cuvettes, tubes or multi-well plates at either room temperature or 37 degrees C. In addition, the reagents are compatible with ELISA assays in which alkaline phosphatase conjugated secondary antibody is used. This kit is well adapted for a number of applications. For example, it can be utilized for direct characterization of enzyme activity and for assay condition optimization. It can also be applied for research diagnosis of diseases that are associated with increased levels of alkaline phosphatase. Typical diseases include liver disease, bone disease, Hodgkin's disease, congestive heart failure, Fanconi's syndrome, hyperparathyroidism, intestinal disease and abdominal bacterial infections. Moreover, it can be used to characterize phosphatase inhibitors through high-throughput screening.\nThe kit reagents have been optimized for long shelf life and maximum reproducibility. The reagents are compatible with all liquid handling systems and bulk reagents are available for high-throughput screening of phosphatase inhibitors.\n\nKey Features:\nHigh sensitivity and wide linear range. The detection limit is generally 3 ng phosphatase or below.\nHomogeneous and simple procedure. No wash or reagent transfer steps are involved. The assay can be completed within 30 minutes.\nRobust and amenable to HTS. All reagents are compatible with high-throughput liquid handling instruments.\n\nApplications:\nEnzyme Activity Assay and Quality Control for phosphatase production. Characterization of Kinetics of phosphatase reaction.\nDrug Discovery: high-throughput screen for phosphatase inhibitors.\n\nKit Contents:\nSize (assays)\nReagent\nAssay Buffer\nStop Solution\n500\nsolid\n25ml\n25ml\n1,000\nsolid\n50ml\n50ml\n2,400\nsolid\n120ml\n120ml\nStorage conditions. The Reagent should be stored in the amber tube at -20 degrees C. The Assay Buffer and Stop Solution are stable at 4 degrees C and room temperature, respectively. Shelf life: 12 months.\n\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

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SPECIFICATIONS

Catalog Number

N2600-36

Size

500Tests

References

Characterization and QC of phosphatases\n1. Roknabadi SM, Bose SK, Taneja V (1999). A histidine thiol 100 kDa, tetrameric acid phosphatase from lentil, Lens esculenta, seeds with the characteristics of protein tyrosine phosphatases. Biochim Biophys Acta. 1433(1-2):272-80.\n2. Wu Q, Gu S, Dai J, Dai J, Wang L, Li Y, Zeng L, Xu J, Ye X, Zhao W, Ji C, Xie Y, Mao Y (2003). Molecular cloning and characterization of a novel dual-specificity phosphatase 18 gene from human fetal brain. Biochim Biophys Acta. 1625(3):296-304.\n3. Umeda IO, Kashiwa Y, Nakata H, Nishigori H (2003). Predominant phosphatase in the ocular lens regulated by physiological concentrations of magnesium and calcium. Life Science 73(9):1161-73.\n4. du Plessis EM, Theron J, Joubert L, Lotter T, Watson TG (2002). Charac-terization of a phosphatase secreted by Staphylococcus aureus strain 154, a new member of the bacterial class C family of nonspecific acid phosphatases. Syst Appl Microbiol. 25(1):21-30.\n5. Ndubuisil MI, Kwok BH, Vervoort J, Koh BD, Elofsson M, Crews CM (2002). Characterization of a novel mammalian phosphatase having sequence similarity to Schizosaccharomyces pombe PHO2 and Saccharomyces cerevisiae PHO13. Biochemistry 41(24):7841-8.\nCharacterization of phosphatase modulators\n6. Urbanek RA, Suchard SJ, Steelman GB, Knappenberger KS, Sygowski LA, Veale CA, Chapdelaine MJ (2001). Potent reversible inhibitors of the protein tyrosine phosphatase CD45. J Med Chem. 44(11): 1777-93.\n7. Santos FT, Scofano HM, Barrabin H, Meyer-Fernandes JR, Mignaco JA (1999). A novel role of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid as an activator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase. Biochemistry 38(32):10552-8.\nELISA assays\n8. Derango R, Page J (1996). The quantitation of coupled bead antibody by enzyme-linked immunosorbent assay. J Immunoassay 17(2): 145-53.\n9. Segal GH, Scott M, Braylan RC (1996). Semi-automated ELISA-based detection system for verifying the authenticity of amplified t(14;18)-containing products. Diagn Mol Pathol. 5(2):114-20.\nClinical diagnostic applications\n10. Delaunay J, Fischer S, Piau JP, Tortolero M, Schapira G (1979). Properties of a membrane-bound phosphatase activity in normal and abnormal red blood cells. Clin Chim Acta. 93(1):15-24.\n11. Spinale FG, Pearce AP, Schulte BA, Crawford FA (1991). Ventricular function and Na+,K(+)-ATPase activity and distribution with chronic supraventricular tachycardia. Cardiovasc Res. 25(2):138-44.\n12. Takeuchi T, Pang M, Amano K, Koide J, Abe T (1997). Reduced protein tyrosine phosphatase (PTPase) activity of CD45 on peripheral blood lymphocytes in patients with systemic lupus erythematosus (SLE). Clin Exp Immunol. 109(1):20-6.\n13. Janckila AJ, Takahashi K, Sun SZ, Yam LT (2001). Naphthol-ASBI phosphate as a preferred substrate for tartrate-resistant acid phosphatase isoform 5b. J Bone Miner Res. 16(4):788-93.\n14. Vihko P, Jokipalo A, Tenhunen R, Alfthan O, Oravisto KJ (1982). Comparison of radioimmunological and conventional acid phosphatase assays in the serum of prostatic cancer patients. Scand J Urol Nephrol. 16: 105-8.\nScreening of phosphatase modulators\n15. Marley AE, Sullivan JE, Carling D, Abbott WM, Smith GJ, Taylor IW, Carey F, Beri RK (1996). Biochemical characterization and deletion analysis of recombinant human protein phosphatase 2C alpha. Biochem J. 320:801-6.