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.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.

H2O: 255 mg/mL (969.4 mM), Sonication is recommended.