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p-Nitrophenyl Phosphate

This p-Nitrophenyl Phosphate is validated for use in ELISA.

Niveau de qualité




Ne contient pas




Conditions de stockage

OK to freeze
protect from light


clearslightly yellow tint

Expédié(e)(s) dans


Temp. de stockage


Description générale

p-Nitrophenyl phosphate is the most common substrate for alkaline phosphatase-based ELISA assays. Hydrolysis of the phosphate ester liberates p-nitrophenol, a soluble, yellow chromogen that can be measured spectrophotometrically at 405 nm (ε = 1.83 x 104 M-1cm-1). As the reaction is linear, kinetic assays can be performed. Endpoint determinations can also be performed by stopping the reaction with strong base and measuring the absorbance at 405 nm. This p-NPP preparation is a stable, convenient, liquid concentrate in a proprietary solvent.
p-Nitrophenyl phosphate supplied as a 50X concentrate (0.19 mol/l). For use with p-NPP Substrate Buffer, Cat. No. 487664.
Excellent substrate for alkaline phosphatase-based ELISA assays. Produces yellow soluble end product that can be read at 405 nm to 410 nm. Both kinetic and endpoint determinations can be performed. Supplied as ready-to-use. A ready-to-use diluent is available separately (Cat. No. 487664).
This p-Nitrophenyl Phosphate is validated for use in ELISA.


10 ml in Glass bottle



Actions biochimiques/physiologiques

Primary Target
Substrate for alkaline phosphatase-based ELISA assays
Product does not compete with ATP.


Toxicity: Standard Handling (A)

Forme physique

Supplied as a 50X concentrate.


Discard if solution turns bright yellow or becomes turbid.

Autres remarques

Functionality check performed on the final product in ELISA.

Suggested procedure for use of p-NPP in alkaline phosphatase-based ELISAs:

1. Prepare 1X p-NPP Substrate Solution: To 39 parts dH2O add 1 part 50X p-NPP Concentrate and 10 parts p-NPP Substrate Buffer (Cat. No. 487664). Mix well and store protected from light. This diluted reagent is suitable for up to 10 h after preparation.

Note: Cat. No. 487664 is an AMP (2-amino-2-methyl-1-propanol)-based buffer. Other buffers, e.g. diethanolamine (DEA)-based and 2-amino-2-methyl-1,3-propanediol (AMPD)-based, may also be employed.

2. Complete all required incubations with antibodies and alkaline phosphatase-labeled probes.

3. Wash plate wells at least 4 times with Tris-buffered saline (TBS) containing 0.1% Tween®-20 detergent. DO NOT USE PHOSPHATE BUFFERS; inorganic phosphate is a potent inhibitor of alkaline phosphatase.

4. After the final wash, shake and blot all residual buffer from the wells.

5. Add 100 µl of 1X p-NPP Substrate Solution (prepared in Step 2) to each well and incubate for 15-60 min. (If a kinetic study is desired, readings at 405 nm may be taken at intermediate intervals).

[Note: Optimal incubation times may vary depending on the amount of enzyme present. If color develops too quickly, dilution of the probe, antibody, or alkaline phosphatase-labeled reagent may be required.]

6. Stop the reaction by adding 100 µl 1 N NaOH.

7. Read absorbance at 405 nm.

Note: Variations in time, reagent volumes, and temperature may require further standardization by the user.

Informations légales

CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany
TWEEN is a registered trademark of Croda International PLC

Code de la classe de stockage

12 - Non Combustible Liquids



Flash Point(F)

Not applicable

Point d'éclair C

Not applicable

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