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p-Nitrophenyl Phosphate (PNPP)

Catalog # Concentration Size List Price Quantity Your Price
P0757L 500 mM 5 ml $225.00
$202.50
P0757S 500 mM 1 ml $58.00
$52.20
Catalog # Size List Price Your Price
P0757L 5 ml $225.00
$202.50
P0757S 1 ml $58.00
$52.20
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

p-Nitrophenyl Phosphate (PNPP) is a non-proteinaceous, non-specific substrate used to assay protein, alkaline and acid phosphatases.

  • Can be used for quick analysis of the protein phosphatase activity under any conditions 
  • Advantageous to a radioactive assay because the substrate concentration can be much higher than the Km
  • 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 that absorbs maximally at 405 nm
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). 
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  p-Nitrophenyl Phosphate (PNPP) P0757SVIAL -20 1 x 1 ml 500 mM
  p-Nitrophenyl Phosphate (PNPP) P0757SVIAL -20 5 x 1 ml 500 mM

Properties & Usage

Molecular Weight

Theoretical: 461.4 daltons


Notes
  • Store protected from light.
  • 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. 
References
  • MacKintosh, C. (1993). In D.G. Hardie (Ed.). Protein Phosphorylation: A Practical Approach. 221. New York: IRL Press.
  • Zhuo, S. et al. (1993). J. Biol. Chem.. 268, 17754-17761.
  • Zhang, Z.-Y. et al. (1992). J. Biol. Chem.. 267, 23759-23766.
  • Pot, D.A. et al. (1991). J. Biol. Chem.. 266, 19688-19696.
  • Takai, A. and Mieskes, G. (1991). Biochem. J.. 275, 233-239.
Quality Control Assay
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
Certificate of Analysis
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Safety Data Sheets
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