Hi Tami, have you tried acid elution wih100 mM glycine, pH2?

Hi Tami, have you tried acid elution wih100 mM glycine, pH2?

Hi there, Two simple explanations : oncolumn aggregation or inaccessibility... I wish you the latter! If HA peptide is not competing it may be due to inaccessibility  to interaction site therefore I would suggest either to move the tag to the Cter or to add a protease cleavage sequence (thrombin, TEV...) between tag and protein in order to elute the adsorbed protein by mild protease treatment. Hope it helps...

Hello, Tami. As Steingrimur suggested, low pH is a very common elution method for antibody based resin. You can also regenerate the column and re-use it in this case. However it will denature your protein. I don't know whether that is of any concern to you, since you tried high pH before, but on the other hand if you do not care about protein being folded you could just as well elute with SDS. It gets somewhat more dirty, but is also more efficient and you get more protein down.

Including a protease site is a great suggestion, unfortunately you would need to re-clone. If you do, do not go for thrombin, it cuts awfully slow and at room temperature. TEV is way better, you could also think of preScission, however that is expensive.

If you stick with the same construct you could use a double purification: His then dialysis then HA and SDS elution (non native). You could do His purification, then possibly ion exchange (if you have fitting pI) and gel filtration (also useful for removing aggregates). Depends on why you are purifying this construct in the first place.

If you will not use HA and stay with the His, batch mode is awfully dirty. If there is AKTA available, it makes great difference. Using overload of the material for very small amount of resin cleans it up. Step elution with increasing imidazole can help to separate contaminants.

Good luck!

Thank you guys for your suggestions!

Unfortunately, keeping the native structure is very much in my interest as I need to use these for enzymatic assays. My trial with increasing the pH was an attempt to see whether I could elute my protein period. I have not tried to do an acid elution with glycine, but it may be worth a shot to see if my protein can be eluted.

Dominique, thanks for the suggestions. I'm guessing my tag may be partially exposed, but it may be I need to try to attach it to the C-terminus. I've been able to use these proteins to do IPs and check for association with other proteins (using the HA tag), but for some reason I really run into problems when I do purification. It may be a wild guess, but perhaps the tag is hidden somehow after elution from the Ni-NTA column. Has anyone every heard of or had something like this happen before?

Maria, I've tested imidazole concentrations and have determined the most stringent conditions but I still seem to have a lot of background as you suggested with a batch purification. We have an FPLC, but I'm afraid my purification may still be too dirty to run through the system. I'll try some of your suggestions though. Thanks a lot!

Hi Tami, you said about the HA column:

"When I've boiled my beads I can see that it comes off easily and most of it is still there"

It could be that the His-tag is obscured by your protein, but it seems that the HA column is working well.

You just have to find the right elution buffer to detach your protein from this antibody affinity column.

I recommend the glycine pH2 buffer. You can collect the eluted material into tubes containing 10X TBS or 10X PBS, so your protein is not in low pH for any amount of time. 

I'm running into this same problem with my HA purification. I am using a StrepIII-HA tag at the C terminus and I can't elute my protein off the HA resin beads using peptide or glycine at pH 2. The only way I've successfully removed the protein from the beads is by boiling in sample buffer.  Did you have any success using urea?