pNPP substrate buffer (diethanolamine, for AP) recipe
Dissolves pNPP substrate for alkaline-phosphatase ELISAs: 1 M diethanolamine, 0.5 mM MgCl₂, pH 9.8; yellow product read at 405 nm.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| Diethanolamine (liquid, density 1.097) | 9.58 mL | 1 M |
| 1 M MgCl₂ | 50 µL | 0.5 mM |
| Water (distilled) | to final volume |
Steps
- Mix diethanolamine with about 70% of the final volume of water (viscous — you can weigh 105.1 g instead).
- Add MgCl₂ and adjust to pH 9.8 with HCl.
- Bring to the final volume with water.
- Store dark at 4 °C.
- Dissolve pNPP at 1 mg/mL just before use, add 100 µL per well and read 405 nm after 15–30 min (stop with 50 µL 3 M NaOH).
Good to know
- Phosphate (PBS) inhibits AP — wash with TBS before AP detection.
⚠ Safety Diethanolamine is irritant — wear gloves and goggles.
FAQ
HRP or AP?
HRP is faster and more common; AP develops linearly over time, making timing easier, and avoids problems with endogenous peroxidase.
This is a widely used standard composition. Amounts depend on the hydrate form and purity of your reagents — check the formula weight (FW) on the bottle and follow your institution's safety rules and the manufacturers' instructions.