TMB substrate solution (homemade) recipe
The ELISA substrate that HRP turns blue — make it just before use: 0.1 mg/mL TMB in phosphate-citrate pH 5.0 with H₂O₂.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| TMB 1 mg/mL (in DMSO) | 1 mL | 0.1 mg/mL |
| 0.05 M phosphate-citrate buffer pH 5.0 | 9 mL | |
| 30% H₂O₂ (add fresh) | 2 µL | ≈0.006% |
Steps
- Dissolve 1 mg TMB in 1 mL DMSO.
- Add to 9 mL phosphate-citrate buffer and mix.
- Just before use add 2 µL 30% H₂O₂ and dispense 100 µL per well.
- Develop 5–30 min in the dark, add an equal volume of stop solution and read 450 nm.
Good to know
- If the solution is already blue, it is contaminated (metal or HRP) — discard it.
- Commercial one-component TMB is more stable with lower background.
⚠ Safety Wear gloves when handling TMB and DMSO.
FAQ
Can azide-containing buffers be used?
No — sodium azide inhibits HRP and kills the signal.
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.