I’m still a little new to this technique [of purifying his-tagged proteins].
I know there are protocols for recharging the columns but does the resin need to be removed before purifying the next protein?
Thank you.
The resin does not need to be removed from the column between purifications. It can be cleaned in place. See the resin manufacturer's instructions for cleaning and recharging the column.
Thank you for your answer.
So even if they are two (or more) different His-tagged proteins, the same column can still be used after recharging it?
You want to be certain that there is no carryover of the first protein into the purification of the second, especially if that would result in some confusion to the results. Stripping the metal from the column and whatever other forms of cleaning that are recommended for the column (such as acid and base washes) will take care of that. All of that should be possible while the resin remains in the column.
Ok. Thank you.
One more question. I’ve heard of adding azide (I’m not sure how much or what concentration) to the column for storage, but does that interfere with the next purification or is that washed out during the purification?
0.02% (w/v) sodium azide prevents growth of bacteria. You can wash it out with buffer before loading the sample onto the column.
Concerning solid sodium azide, be careful when handling this toxic and potentially explosive compound.
Ok.
Yes, azide is pretty nasty stuff (binds cyt C, forms explosive nitrides with metals).
The protocol mentions the EDTA in the charging steps prevents bacterial growth too, which is washed out during the next purification, so I wasn't sure if azide was better to use or not.
I also was not sure regarding my initial question about adding new resin with another purification, because the protocol says to use new resin with each new protein purified.
Anyway, thank you so much for your answers. I feel better now about moving forward with this.
EDTA strips the metal ions from the resin. It should be washed out before recharging the column.
For small columns, it may be more efficient to use them once and discard them. For larger columns, the cost of the resin or pre-packed column may make it worth the effort to clean them.
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