p-Nitrophenyl Phosphate Substrate Buffer

Alkaline phosphatase substrate buffer supplied as ready-to-use. A diluent for Cat. No. 487663.

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

ELISA (see comments)

Primary Target
Alkaline phosphatase substrate buffer

Product does not compete with ATP.

Toxicity: Standard Handling (A)

Product tested for stability at 4°C, 18-26°C, and 37°C. Product performance is tested on the final product by ELISA.



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



1. To 39 parts distilled water add 1 part 50X p-NPP Concentrate (Cat. No. 487663) and 10 parts p-NPP Substrate Buffer. Mix well and store protected from light. This diluted reagent is suitable for up to 10 h after preparation.

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 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 phophatase-labeled reagent may be required.]

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

7. Read absorbance at 405 nm.



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

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

TWEEN is a registered trademark of Croda International PLC