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487663

Sigma-Aldrich

p-Nitrophenyl Phosphate

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

Quality Level

form

liquid

does not contain

preservative

manufacturer/tradename

Calbiochem®

storage condition

OK to freeze
protect from light

color

clearslightly yellow tint

shipped in

ambient

storage temp.

2-8°C

General description

This p-Nitrophenyl Phosphate is validated for use in ELISA.
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.
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).
p-Nitrophenyl phosphate supplied as a 50X concentrate (0.19 mol/l). For use with p-NPP Substrate Buffer, Cat. No. 487664.

Packaging

10 ml in Glass bottle

Application

ELISA

Biochem/physiol Actions

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

Warning

Toxicity: Standard Handling (A)

Physical form

Supplied as a 50X concentrate.

Reconstitution

Discard if solution turns bright yellow or becomes turbid.

Other Notes

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.

Legal Information

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

Storage Class Code

12 - Non Combustible Liquids

WGK

WGK 3

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Certificate of Analysis

Certificate of Origin

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