HisTrap / His-Tag IMAC Buffer Calculator
Scale method-specific HisTrap binding and elution buffers containing 20 mM Tris-HCl, 300 mM NaCl, and fixed imidazole concentrations.
Select a reviewed preset and enter the final volume to prepare.
| Reagent | Formula | Amount or action |
|---|---|---|
| 1 M Tris-HCl pH 7.4prepared aqueous stock | 20 mL | |
| Sodium chlorideanhydrous | 17.5 g | |
| Imidazolefree base | 2.7 g | |
| pH adjustment reagent (unspecified) | Measure the final mixture at 25 °C and adjust to pH 7.4; the adjustment reagent/titrant is unspecified by the source. | |
| Purified water | After pH adjustment, bring the total solution volume to 1 L. Do not add 1 L of water; add water only until the total solution reaches 1 L. |
How to prepare HisTrap binding buffer
- Select the binding or elution preset and add the calculated 1 M Tris-HCl pH 7.4 stock volume to less than the selected final volume of purified water.
- Add the calculated NaCl and imidazole amounts for the selected preset.
- Stir until all solids dissolve completely.
- Measure the final mixture at 25 °C and adjust to pH 7.4.
- Bring to the selected final volume with purified water, mix thoroughly, and verify the final pH.
- Filter the completed buffer through a 0.22 µm membrane.
About This Buffer
Immobilized metal affinity chromatography (IMAC) is commonly used to purify histidine-tagged proteins, with imidazole used to control binding and elution conditions. Exact buffer compositions depend on the column, resin, and purification method. The ChemH calculator therefore provides one method-specific HisTrap system: a binding buffer containing 20 mM Tris, 300 mM NaCl, and 40 mM imidazole and an elution buffer containing 20 mM Tris, 300 mM NaCl, and 500 mM imidazole. Both are adjusted to pH 7.4 at 25 °C using an unspecified titrant, as defined by the reviewed method.
Common questions
What is the difference between the binding and elution presets?
Both contain 20 mM Tris-HCl and 300 mM NaCl. The binding buffer contains 40 mM imidazole, while the elution buffer contains 500 mM imidazole.
Are these universal Ni-NTA recipes?
No. They are method-specific HisTrap formulations and should not be presented as universal conditions for every nickel-affinity resin.
Why is the page named HisTrap rather than Ni-NTA?
The reviewed source describes HisTrap conditions. HisTrap uses a specific nickel-affinity medium and should not be treated as identical to every Ni-NTA product.
Sources and review notes
The source for the currently selected preset appears first. Recipe quantities remain tied to the cited article location.
HisTrap binding buffer
Source location: Buffer preparation — HisTrap binding buffer, 20 mM Tris-HCl, 300 mM NaCl, 40 mM imidazole, final pH 7.4 at 25 °C; filter the completed buffer through a 0.22 µm membrane.
Preparation method: Source-stated quantitative composition
- Preparation of scFv stabilized chromatosomes for single-particle cryo-EM structure determination PMC7994535 · open article
View sources for all presets
HisTrap elution buffer
Source location: Buffer preparation — HisTrap elution buffer, 20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, final pH 7.4 at 25 °C; filter the completed buffer through a 0.22 µm membrane.
Preparation method: Source-stated quantitative composition
- Preparation of scFv stabilized chromatosomes for single-particle cryo-EM structure determination PMC7994535 · open article