I'm purifying a homodimer that is coexpressing subunits with different tags to be able to mutate subunits independently from eachother. One subunit has a His tag and the other a FLAG tag. My question is whether order of purification matters. I'd like to use the Ni-nta column first (eluting with 100mM imidazole) followed by the M2 FLAG resin (eluting with low pH). Can anyone think of a reason why this shouldn't work before I get too far into this? All the papers I've read seem to purify FLAG first followed by His when doing TAP, but I haven't found a good reason for this and I'm not sure if what I'm doing is really TAP. Thanks!!
It looks like your strategy is correct. Of course, one of the tags might not be easily accessible for binding (needs to be tested), but since your protein is a homodimer you selected the right way to go. Eluting with low pH might cause the subunit dissociation (and you might want to avoid that), because the next step of purification might generate only one subunit (not the homodimer).
I also recommend using His-NTA first because it is less specific and therefore dirtier. A subsequent purification with FLAG will not only help improving purity but also protect your samples like you have suggested. Also, this way you will get rid of excess imidazole that you might use for elution from Ni beads.
Hi Jessica,
Your strategy seems OK, especially if you elute from the M2 FLAG resin at low pH: the low pH would reduce the subsequent binding to the Ni-NTA. But if this does not work well, you can still try the other way round, by using a buffer exchange column or a dialysis step before loading your sample on the Ni-NTA.
I do not know if it applies here, but in general, you use first a high capacity, cheaper resin (because the volume of the sample at that step will be large) and then a lower capacity, possibly more expensive, more resolutive column, because at that step, the sample will be concentrated in a smaller volume and partially purified (and so you have less risk to clog the column).
In theory it shouldn't matter so you should go for the column with bigger capacity as first. However, what Viktor and Kshitiz wrote are valid points (dissociation in acidic pH and removal of imidazole).
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