Application

ELISA

Biochem/physiol Actions

Product does not compete with ATP.

Primary TargetSubstrate for alkaline phosphatase-based ELISA assays

General description

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 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.

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

p-Nitrophenyl phosphate supplied as a 50X concentrate (0.19 mol/l). For use with p-NPP Substrate Buffer, Cat. No. 487664.

Legal Information

TWEEN is a registered trademark of Croda International PLC

CALBIOCHEM is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Functionality check performed on the final product in ELISA.r>r>Suggested procedure for use of p-NPP in alkaline phosphatase-based ELISAs:r>r>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.r>r>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.r>r>2. Complete all required incubations with antibodies and alkaline phosphatase-labeled probes.r>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.r>4. After the final wash, shake and blot all residual buffer from the wells. r>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).r>r>[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.]r>r>6. Stop the reaction by adding 100 µl 1 N NaOH. r>7. Read absorbance at 405 nm.r>r>Note: Variations in time, reagent volumes, and temperature may require further standardization by the user.

Packaging

10 ml in Glass bottle

Physical form

Supplied as a 50X concentrate.

Reconstitution

Discard if solution turns bright yellow or becomes turbid.

Warning

Toxicity: Standard Handling (A)