pNPP-Na hexahydrate, Alkaline phosphatase substrate (ab146203)


  • Product name

    pNPP-Na hexahydrate, Alkaline phosphatase substrate
  • Description

    Alkaline phosphatase substrate.
  • Alternative names

    • 4-Nitrophenyl phosphate disodium salt hexahydrate
    • Disodium 4-nitrophenyl phosphate hexahydrate
    • p-Nitrophenyl phosphate disodium hexahydrate
    • pNPP disodium hexahydrate
    • pNPP-2Na hexahydrate
    • pNPP-Na
    see all
  • Biological description

    Chromogenic substrate for acid and alkaline phosphatases. The reaction produces a soluble end product that is yellow in color. The reaction may be stopped with 3 N NaOH and read at 405 nm. Can be used to perform kinetic and endpoint determinations.

  • Purity

    > 98%
  • CAS Number

  • Chemical structure

    Chemical Structure


  • Molecular weight

  • Molecular formula

  • Storage instructions

    Store at -20°C. Store under desiccating conditions. The product can be stored for up to 12 months.
  • Handling

    Wherever possible, you should prepare and use solutions on the same day. However, if you need to make up stock solutions in advance, we recommend that you store the solution as aliquots in tightly sealed vials at -20°C. Generally, these will be useable for up to one month. Before use, and prior to opening the vial we recommend that you allow your product to equilibrate to room temperature for at least 1 hour.

    Need more advice on solubility, usage and handling? Please visit our frequently asked questions (FAQ) page for more details.

  • Source



This product has been referenced in:

Customer reviews and Q&As


I can confirm that ab146203 (pNPP-Na hexahydrate) is the correct substrate for Alkaline Phosphotase (coupled to an antibody).

pNPP is hydrolyzed rapidly in the presence of Alkaline Phosphatase to p-nitrophenol and inorganic phosphate.

The formed p-nitrophenol has an intense yellow color that is measured at 405 nm. Making pNPP reagents for AP assays involves preparing a stock solution, that is stable for limited time, and dilution to final using concentration, that is stable for one day only.

CAUTION: Avoid pNPP contact with eyes, skin, and clothing. Please see the MSDS for handling information ( can be downloaded from the datasheet).

1. Equilibrate pNPP to room temperature before opening.

2. Weigh desired amount (powder) as needed – i.e. weigh 5mg powder

3. Dissolve 5mg of pNPP in 5mL of suitable substrate buffer.

Note: a standard assay buffer is 100 mM Tris-HCl, pH 9.5 , 100 mM NaCl , 5 mM MgCl2.

Alternative buffers have been used:

-Diethanolamine solution at 10mM to 1M: 5.1M DEA, pH 9.8 ; or10mM DEA 0.5M Magnesium Chloride, pH9.5

-in cell preparations containing AP: 10 mM pNPP and 2 mM EDTA in 20 mM MES at pH 6.0

A standard concentration is 1mg pNPP/mL solution, but this may be optimized in your application.

Note: This working solution should be prepared just before use. Protect from light. Discard unused reagent.

For standard ELISA

4. Add 100μL of the PNPP solution to each microplate well.

Incubate plate at at 37°C for 30 minutes or until sufficient color develops.

Note: room temperature incubation also works. It is thought to give less border signal variation, but weaker signal and less reproducible results from

experiment to experiment.

5. (optional – end point assays) To stop the reaction, add 50μL of 2N NaOH to each well.

6. Measure the absorbance at 405nm with a standard microplate reader.

-In end-point assays, pNPP incubation time may be optimized between 10 and 60 min. Wells are added or not with

100μl reaction stop solution (i.e. 1 N NaOH with 0.25 M EDTA ), and color is measured at 405 nm.

-In kinetic assays, the formed yellow color appears rapidly and monitored in a kinetic ELISA reader at 405/650 nm.

Read More

For licensing inquiries, please contact

Sign up