DESCRIPTION
Para-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.
                p-Nitrophenyl phosphate -------phosphatase ------>p-nitrophenol + phosphate
This 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°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.
The 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.

KEY FEATURES
High sensitivity and wide linear range. The detection limit is generally 3 ng phosphatase or below.
Homogeneous and simple procedure. No wash or reagent transfer steps are involved. The assay can be completed within 30 minutes.
Robust and amenable to HTS. All reagents are compatible with highthroughput liquid handling instruments.

APPLICATIONS
Enzyme Activity Assay and Quality Control for phosphatase production.
Characterization of Kinetics of phosphatase reaction.
Drug Discovery: high-throughput screen for phosphatase inhibitors.

KIT CONTENTS
Catalog #      Size (assays)      Reagent        Assay Buffer      Stop Solution
POPN-500         500               solid                 25 mL                 25 mL
POPN-01K      1,000              solid                 50 mL                 50 mL
POPN-2.4K     2,400              solid                120 mL               120 mL
POPN-HTS       >10k              solid              customized        customized
Storage conditions. The Reagent should be stored in the amber tube at - 20°C. The Assay Buffer and Stop Solution are stable at 4°C and room temperature, respectively. Shelf life: 12 months.

Precautions: 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.

PROCEDURES
Procedure using 96-well plate:
          1. Equilibrate Assay Buffer and Stop Solution to room temperature by allowing them to stand for 30 minutes at room temperature. Reconstitute the Reagent with the provided Assay Buffer. Simply combine the Assay Buffer and Reagent by pipetting a small volume (e.g. 1 mL) buffer to the
Reagent tube. Vortex briefly and transfer the reconstituted solution to the Assay Buffer bottle. Repeat this step to transfer all Reagent to the Assay Buffer bottle. Mix by inversion until the Reagent is thoroughly dissolved.
After this is done, mark the bottle label as pNPP Substrate.
          2. Series dilute enzyme in a proper Enzyme Buffer. Prepare enough solution for triplicate assays. Transfer 50 µL of each enzyme dilution to wells in the test plate. In addition, prepare blank wells that contain 50 µL Enzyme Buffer without any enzyme. Initiate the reaction by adding 50 µL
pNPP Substrate.
          3. Incubate for 10-30 minutes at room temperature.
          4. Stop the reaction by adding 50 µL Stop Solution. Mix by quickly tapping the plate. Alternatively, plates can be shaken for 10 seconds on an orbital plate shaker.
          5. Read the absorbance of each well at 405 nm.

Procedure using 384-well plate:
          1. Equilibrate Assay Buffer and Stop Solution to room temperature by allowing them to stand for 30 minutes at room temperature. Reconstitute the Reagent with the provided Assay Buffer. Simply combine the Assay Buffer and Reagent by pipetting a small volume (e.g. 1 mL) buffer to the
Reagent tube. Vortex briefly and transfer the reconstituted solution to the Assay Buffer bottle. Repeat this step to transfer all Reagent to the Assay Buffer bottle. Mix by inversion until the Reagent is thoroughly dissolved.
After this is done, mark the bottle label as pNPP Substrate.
          2. Series dilute enzyme in a proper Enzyme Buffer. Prepare enough solution for triplicate assays. Transfer 25 µL of each enzyme dilution to wells in the test plate. In addition, prepare blank wells that contain 25 µL Enzyme Buffer without any enzyme. Initiate the reaction by adding 25 µL
pNPP Substrate.
          3. Incubate for 10-30 minutes at room temperature.
          4. Stop reaction by adding 25 µL Stop Solution. Mix by quickly tapping the plate. Alternatively plates can be shaken for 10 seconds on an orbital plate shaker.
          5. Read the absorbance of each well at 405 nm.

GENERAL CONSIDERATIONS
          (1). Fresh reconstitution of the Reagent is recommended although the reconstituted pNPP Substrate may be stable for up to 4 weeks when stored at -20 °C. (2). Assays can be performed at room temperature or at 37 °C. (3). The pH of the Assay Buffer is 7.2 and is compatible with the majority of phosphatases. For an acid phosphatase, we recommend using 100mM sodium acetate (pH 5.5), 10mM MgCl2 as Enzyme Buffer.
For an alkaline phosphatase, we recommend using 100mM Tris-HCl (pH 8.6), 10mM MgCl2 as Enzyme Buffer.

DATA ANALYSIS
Calculate the average and standard derivations of the triplicate assays and subtract the blank values. Enzyme activity is calculated from Beer- Lambert law as follows, where e is the molar extinction coefficient (M-1 × cm-1). For p-nitrophenol, e = 1.78 x 104 M-1 × cm-1. OD405nm (cm-1) is the absorbance at 405 nm divided by the light-path length (cm). V is the final assay volume, i.e., 150
µL for 96-well plate assay and 75 µL for 384-well plate assay.

LITERATURE
1. Monick, M.M. et al (2006). Active ERK Contributes to Protein Translation by Preventing JNK-Dependent Inhibition of Protein Phosphatase. J. Immunol. 177: 1636–1645.
2. Nakano, Y. (2007). Novel function of DUSP14/MKP6 (dual specific phosphatase 14) as a nonspecific regulatory molecule for delayed-type Enzyme activity (µmoles/min/µg) =
V (µL) x OD405nm (cm-1) e x incubation time (min) x enzyme (µg) Enzyme turn-over number (min-1) = Enzyme activity (µmoles/min/µg) x enzyme molecular weight (dalton) hypersensitivity. British J. Dermatology 156 (5): 848–860.
3. Lee, S.W. et al (2008). The Xanthomonas oryzae pv. oryzae PhoPQ twocomponent system is required for AvrXA21 activity, hrpG expression, and virulence. J. Bacteriol.190(6):2183-97.

Characterization and QC of phosphatases
           1. 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.

          2. 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.
           3. 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.
           4. 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.
           5. 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. Characterization of phosphatase modulators
          6. 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.
7. 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.
ELISA assays
8. Derango R, Page J (1996). The quantitation of coupled bead antibody by
enzyme-linked immunosorbent assay. J Immunoassay 17(2): 145-53.
9. 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.
Clinical diagnostic applications
10. 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.
11. 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.
12. 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.
13. 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.
14. 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.
Screening of phosphatase modulators
16. 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.

TECHNICAL NOTES
The pNPP assay kits have been specially optimized and formulated to
provide a convenient and sensitive assay for a large number of
phosphatases. Key features of the kits are as follows:
Good sensitivity and wide linear range. The detection limit is generally
3 ng phosphatase or below.
Homogeneous and simple procedure. No wash or reagent transfer
steps are involved. The assay can be completed within 30 minutes.
Robust and amenable to HTS. All reagents are compatible with highthroughput
liquid handling instruments.