His-tag elution buffer (250 mM imidazole) recipe
Elutes His-tagged protein from Ni-NTA: 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, pH 8.0.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| NaH₂PO₄·H₂O | 3.45 g | 50 mM |
| NaCl | 8.77 g | 300 mM |
| Imidazole | 8.51 g | 250 mM |
| Water (distilled) | to final volume |
Steps
- Dissolve everything in about 80% of the final volume of water.
- Adjust to pH 8.0 with NaOH (after adding imidazole, which shifts the pH).
- Bring to the final volume with water.
- Filter and store at 4 °C.
Good to know
- The Ni-NTA trio: lysis 10 mM → wash 20 mM → elution 250 mM imidazole. Raise the wash to 20–40 mM if non-specific binding is high.
- Avoid EDTA and DTT, which strip nickel (use ≤20 mM β-ME if a reductant is needed).
FAQ
Does imidazole interfere with A280?
High imidazole absorbs at 280 nm — blank with the same buffer or quantify by BCA/Bradford.
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.