A polyhistidine tag (generally 5 or more His residues in a row) added to either the N or C terminus will chelate nickel or cobalt ions with high affinity. When the pH is decreased to 3-4, the imidazole group on histidine becomes protonated and the protein is eluted from the metal ions. This system has the advantage over other affinity purification techniques that it does not require the tag or protein to be properly folded, as binding is due only to the primary protein structure. Denaturing conditions (such as those required to solublise inclusion bodies) can therefore be used during the purification process.
Preparing the column
- When taking resin out of storage, the resin is likely to have precipitated from its storage buffer. Resuspend the resin in its bottle by inverting and gently tapping the bottle repeatedly.
- Pipette 2 ml of the resin into a 10-ml affinity column. Allow the resin to settle completely by gravity (5-10 minutes) before carefully aspirating the supernatant.
- Add 6 ml of sterile, distilled water and resuspend the resin by alternately inverting and gently tapping the column.
- Allow the resin to settle again using gravity, and gently aspirate the supernatant.
- Add 6 ml of Denaturing Binding Buffer and resuspend the resin by alternately inverting and gently tapping the column.
- Allow the resin to settle using gravity, and gently aspirate the supernatant. Repeat Steps 5 and 6.
- Clamp a prepared purification column vertically at a convenient height for pipetting into. Add 8 ml lysate from an [inclusion body prep].
- Bind for 15–30 minutes at room temperatureby inverting up and down to keep the resin suspended in the lysate solution. Settle the resin by gravity and carefully aspirate the supernatant.
- Wash the column with 4 ml Denaturing Binding Buffer by resuspending the resin and rocking for two minutes. Settle the resin by gravity, and carefully aspirate the supernatant. Save supernatant at 4°C so it can be analysed later for diagnostic purposes. Repeat this step one more time.
- Wash the column with 4 ml Denaturing Wash Buffer, pH 6.0 by resuspending the resin and rocking for two minutes. Settle the resin by gravity, and carefully aspirate the supernatant. Save supernatant at 4°C. Repeat this step one more time.
- Wash the column with 4 ml Denaturing Wash Buffer pH 5.3 by resuspending the resin and rocking for 2 minutes. Settle the resin by gravity, and carefully aspirate the supernatant. Save supernatant at 4°C. Repeat this step once more.
- Snap off the cap on the lower end. Elute the protein by adding 5 ml Denaturing Elution Buffer. Collect 1 ml fractions and monitor the elution by taking OD280 readings of the fractions. Pool the fractions that contain the peak absorbance and dialyze against 10 mM Tris, pH 8.0, 0.1% Triton X-100 overnight at 4°C to remove the urea. The reflectin should precipitate in the tubing.
The first time we attempted this protocol, we achieved a relatively low yield which we believed could be vastly improved by adopting the following techniques to avoid accidentally removing resinous material when aspirating the supernatant from the column:
- Use a low volume pipette (max. 1ml), particularly when aspirating the supernatant from the his-trap column. This reduces the suction power of the pipette.
- Wait until the resin has completely settled before aspirating the supernatant fluid; we learned that this happens only when the supernatant is entirely transparent (with no cloudiness).
- Ensure that the pipette tip is at least 2mm above the level of the resin when aspirating.
- Do not try to remove every last drop of supernatant; it is more important to avoid accidentally removing the resin. Leaving 1mm of liquid above the resin works fine.
Alternatively, allowing the wash buffers to run through rather than pipetting off seems to give similar yield with no resin loss.
All safety measures relating to the use of the buffers are detailed in their protocol. In addition, all consumables and liquid waste that may have come into contact with the bacteria must be autoclaved before disposal.
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