Affinity Chromatography - Department of Molecular and Cellular ...

Nickel solution: 0.1 M NiSO 4
Binding buffer: 20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, pH 7.4
Elution buffer: 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4
1. Wash the column with 5 column volumes of distilled water.
Use water, not buffer, to wash away the column storage solution which contains 20% ethanol.
This avoids the risk of nickel salt precipitation in the next step. If air is trapped in the
column, wash the column with distilled water until the air disappears.
2. Load 0.5 column volumes of the 0.1 M nickel solution onto the column.
3. Wash with 5 column volumes of distilled water.
4. Equilibrate the column with 10 column volumes of binding buffer.
5. Apply sample at a flow rate 1–4 ml/min (1 ml column) or 5 ml/min (5 ml column). Collect the flow-through
fraction. A pump is more suitable for application of sample volumes greater than 15 ml.
6. Wash with 10 column volumes of binding buffer. Collect wash fraction.
7. Elute with 5 column volumes of elution buffer. Collect eluted fractions in small fractions such as 1 ml to avoid
dilution of the eluate.
8. Wash with 10 column volumes of binding buffer. The column is now ready for a new purification and there is
rarely a need to reload with metal if the same (His) 6 fusion protein is to be purified.
Imidazole absorbs at 280 nm. Use elution buffer as blank when monitoring absorbance.
If imidazole needs to be removed, use a desalting column (see page 134).
For a single purification of a small quantity of product or for high throughput screening
His MicroSpin columns are convenient and simple to use with either centrifugation or
MicroPlex 24 Vacuum.
To increase capacity use several HiTrap Chelating HP columns (1 ml or 5 ml) in series.
HiTrap Chelating HP columns (1 ml or 5 ml) can be used with a syringe, a peristaltic pump
or a chromatography system. For even larger capacity, pack Chelating Sepharose Fast Flow
into a suitable column (see Appendix 3).
The loss of metal ions is more pronounced at lower pH. The column does not have to be
stripped (i.e. all metal ions removed) between each purification if the same protein is going
to be purified. In this case, strip and re-charge (i.e. replace metal ions) the column after
5–10 purifications.
Reuse of purification columns depends on the nature of the sample and should only be
performed with identical samples to prevent cross contamination.
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