His-tag purification buffers (lysis, wash, elution) recipe
The three buffers for purifying His-tagged proteins on Ni-NTA. All are 50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0 and differ only in imidazole: lysis 10 mM → wash 20 mM → elution 250 mM.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| NaH₂PO₄·H₂O | 3.45 g | 50 mM |
| NaCl | 8.77 g | 300 mM |
| Imidazole | 340 mg | 10 mM |
| NaH₂PO₄·H₂O | 3.45 g | 50 mM |
| NaCl | 8.77 g | 300 mM |
| Imidazole | 680 mg | 20 mM |
| 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
- Order of use: lyse and bind in lysis buffer → wash with 10–20 resin volumes of wash buffer → elute with elution buffer.
- If non-specific binding is high, raise wash imidazole to 20–40 mM; lower it if your protein comes off in the wash.
- 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.
How do I remove imidazole after elution?
Dialyse or exchange the buffer on a desalting column. Do it soon after elution, as some proteins aggregate in high imidazole.
References
- Hochuli E, Bannwarth W, Döbeli H, Gentz R, Stüber D. Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Bio/Technology. 1988;6:1321–1325.
- Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor Laboratory Press; 2012.
- Harlow E, Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press; 1988.
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.