PNPP is a non-proteinaceous chromogenic substrate for alkaline and acid phosphatases used in ELISA and conventional spectrophotometric assays.

Instructions
I. Solution preparation
1. Stock solution preparation: Dissolve PNPP powder in an appropriate buffer, usually at a concentration of 5–10 mM. It is recommended to use a buffer containing 50 mM Tris-HCl and 10 mM MgCl₂, with the pH adjusted to 9.5 to suit the activity of alkaline phosphatase.
2. Working solution preparation: Dilute the stock solution to the required concentration, usually 2–5 mM, according to experimental needs.
II. Operation steps
1. Reaction conditions:
Temperature: 37°C. Time: 10–30 minutes, the specific time needs to be optimized according to the experiment.
2. Detection method:
1) Under the action of the enzyme, PNPP is hydrolyzed to produce yellow p-nitrophenol, which has a maximum absorption peak at 405 nm.
2) Use a spectrophotometer to measure the absorbance at 405 nm to evaluate the activity of the enzyme.
Note:
Fresh preparation: PNPP solution should be prepared before use and avoid long-term storage to prevent hydrolysis.
Store away from light: PNPP is light-sensitive, so strong light exposure should be avoided during operation.
Termination of reaction: In ELISA experiments, an equal volume of 0.1 M NaOH or 0.1 M EDTA can be added after the reaction is completed to terminate the reaction.