I'm currently fighting with the Strep-tag/Strep-tactin chromatography.
I observed, that the HABA is not eluted completely even by 100 mM Tris base (pH ~10.5). After the loading of proteins, I elute with 2.5 mM desthiobiotin, then I apply 1 mM HABA (purchased as 10x* solution, arounf 15 CV) and then wash with 100 mM Tris base (about 10 CV). When I apply directly desthiobiotin to such treated column, I see very nice peak of HABA in the chromatogram. All steps are performed at 3 ml/min (recommended flow rate for 5 ml column) at 4°C. Does anybody of you have such experience? What could be possibly done to remove it?
GE Healthcare suggests use of 0.5 M NaOH for regeneration instead of HABA, but IBA, from which we have the column, do not even mention that. Could it be because they would get no money for NaOH or because it is not so good for the Strep-Tactin?
Also, I have no binding of my protein to the column. I tried decreased flowrate and pause while is the sample on column, but didn't work (could be because of the HABA though). I have confirmed presence of the Strep-tag II by antibody yesterday. What other parameters there are one can change to improve binding to Strep-Tactin column?
EDIT fixed *10x, I haven't pressed Shift while writing numbers on Czech keyboard :)
I experienced similar things with HABA regeneration, but with buffers around pH 8. You did not mention how the column looked like. In my experience it took a lot of CVs to get the column perfectly colorless again, maybe 10 CV is not enough. Also make sure that you do the washing really thorough without any HABA buffer sticking around somewhere in the column in case you use a self-packed gravity flow column.
concerning the binding you might want to switch the tag to the other terminus of the protein and maybe increase the linker between protein and tag.
we have been using a double strep with a linker this has significantly improved binding to the strep resin (we are now using the high capacity IBA resin which is a real improvment on the Qiagen 1ml cartridge we previously used), only problem we have had is elution, although we now elute with 10mM Desthiobiotin with a paused elution (60mins), we also dont bother re-using columns or resin we just take a small amount of fresh resin (~1ml) and re-pack
I have generally had good experiences with the StrepII tag and StrepTactin column. I agree with both of the above answers. I generally wash with 20CV of Tris/EDTA buffer pH 8 (wash buffer in the IBA protocol). I use Qiagen FPLC cartridges, and the column never gets completely colorless, but works fine for subsequent purifications.
You should be able to use the GE regeneration protocol for the IBA resin.
As for your protein not binding, it is possible your tag is not exposed in solution. As mentioned above, try switching the position of the tag (I have done that and it helped) or adding a linker. Have other tags been used successfully in the orientation you have used the StrepTag in?
Hi,
thanks for your answers.
Concerning the HABA removal... Well, it turned out to be rather my fault. Since I was using four different solutions I used all the lines, but I didn't purge it before run because I wanted to save the elution buffer and I didn't realize there's the HABA in between the pump and the mixer. I'll try it again, probably next week and I'll see whether that was the problem.
myc-tag was used previously on the C-terminus as well and it was succesful with purification. The problem is, that I tried other protein, which worked previously with StrepTactin in batch method and in my hands it didn't work (I'm repeating the PAGE and bloting, because it didn't work very well). So I'll have to try it the batch methods first time.
Good luck. By the way, what kind of protein are you trying to purify? Cytoplasmic? Does it have properties that would make it aggregate/precipitate out? I assume you clarify your lysates by centrifugation prior to loading on the column. Is it possible your protein is in the pellet fraction? Do you run out input, flowthrough, washes and elution fractions on SDS-PAGE and track your protein by western blot through the purification process?
Well, the localization seems to be little tricky, it's probably associated with something like cell wall maybe... (unextractable with 1% Triton), but the part I'm working with is soluble protein.
I put everything on the western (or when I measure activity) and it's clearly in the unbound while nothing in elution.
If all it is is HABA in your lines, hopefully a more thorough wash will solve the issue. If you are still unable to see binding, you may need to think about switching the tag to the other terminus or adding a linker. Maybe you'll have better luck with batch purification.
Yeah, unfortunately I tried batch purification with the MacroPrep yesterday and it didn't work :-/ Now I'm doing blot of the positive controls, so I'll see if it's me or just the protein...
In my experience, the problem of binding of the tag to the Strep-Tactin column was solved after introducing a linker between the Strep-II tag and the C-terminus of the protein in question. Thorough washing between different steps and during the regeneration has always primary importance.
Hi Tomas,
If you have the problem still now with protein binding to column then...check the protein sample pH before loading to the column. I don't know what type of buffer and pH of buffer you are using for the protein extraction. I faced similar problem with my protein. Then I adjusted the pH (final pH 8 ) of the crude protein sample adding some drops of dilute NaOH. I always adjust the protein sample pH 8 as I am using binding buffer and Elution buffer with pH 8. In my protein the tag is at the N-terminal end and its seems ok. The regeneration of column is nice with 0.5 M NaOH....
The protein was re-cloned in the meanwhile with GFP in between my protein and the Strep-tag and this construct worked quite nicely. We stil had some contaminating proteins though.
To get rid of HABA, I always wash the column with 0.5M NaOH and then quickly equilibrate it with Tris-NaCl buffer, it works completely fine.
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