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  • p-Nitrophenyl Phosphate (PNPP)


    p-Nitrophenyl Phosphate (PNPP) is a non-proteinaceous, non-specific substrate used to assay protein, alkaline and acid phosphatases. The PNPP phosphatase activity is measured using a continuous or single-point spectrophotometric assay based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol, a chromogenic product with absorbance at 405 nm (1). The assay can be used for the quick analysis of the protein phosphatase activity under any non-standard conditions. 

    The advantage of the PNPP phosphatase activity assay is that unlike radioactive assays the substrate concentration can be much higher than the Km. The initial velocity can be recorded in the continuous assay, but the assay volume is larger than in a radioactive assay (about 1 ml to fill a 1 ml spetrophotometer cuvette) (1,2). The reaction volume in a single-point assay can be very small because the reaction is stopped with the amount of NaOH enough to fill the cuvette (1,3,4). 

    Properties and Usage

    Storage Temperature


    Molecular Weight

    Theoretical: 461.4 daltons


    1. Store protected from light.
    2. Protein Phosphatase Assay: The PNPP phosphatase activity assay is very simple, non-expensive, and routinely used for the unit determination of all NEB protein phosphatases. PNPP has apparent Km values for protein phosphatases in the range of 0.5 -10 mM (2-5).

      The PNPP phosphatase activity is assayed in a reaction mixture (50 μl) containing 50 mM PNPP and a protein phosphatase buffer supplemented with additional components when required. The reaction is initiated by addition of enzyme and quenched after 5-10 minutes by addition of 1 ml of 1 N NaOH (or 1 ml of 0.5 M EDTA for Mn2+-dependent protein phosphatases, Lambda PP and PP1). The amount of product, p-nitrophenol, is determined by reading the absorbance at 405 nm and using a molar extinction coefficient of 18,000 M-1 cm-1 (16,000 M-1cm-1 for 0.5 M EDTA) (1, 3). 

      One unit of the protein phosphatase activity is defined as the amount of enzyme that hydrolyzes 1 nanomole of PNPP in one minute at 30°C in a total reaction volume of 50 μl under standard reaction condition. 

      To estimate the protein phosphatase activity accurately it is essential to ensure linear kinetics of dephosphorylation. 


    1. MacKintosh, C. (1993). In D.G. Hardie (Ed.). Protein Phosphorylation: A Practical Approach. 221. New York: IRL Press.
    2. Zhuo, S. et al. (1993). J. Biol. Chem.. 268, 17754-17761.
    3. Zhang, Z.-Y. et al. (1992). J. Biol. Chem.. 267, 23759-23766.
    4. Pot, D.A. et al. (1991). J. Biol. Chem.. 266, 19688-19696.
    5. Takai, A. and Mieskes, G. (1991). Biochem. J.. 275, 233-239.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.


    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.