I feel that this method is not as straightforward as it is described by IBA and Co. I did not detect much useful information on the web, except for the manuals provided by the commercial suppliers. I am currently trying to purify a membrane protein with the strep tag on the Cterminal from which I know that it is on a small cytoplasmic domain. A homologous X-ray structure exists suggesting that the tag is accessible.
My problems:
1. Strep Tactin is not really compatible with many detergents (especially short chain, DM, DPC, ....) at "reasonable" concentrations (> 2 cmc) - in contradiction to the statement from IBA suggesting application of this method for the purification of membrane proteins. Therefore, this type of purification limits the choice of detergent.
2. In DDM micelles, only ca 50% of my protein is retained by the column (100 cmc DDM, 100 mM TRIS pH 8.0, 150 mM NaCl, 1 mM EDTA, 10% glycerol). Washing and elution are performed in the same buffer but at 10 cmc DDM and without glycerol. Western blot (specific detection of tagged protein) shows that the protein is in the flow-through (no proteolysis observed).
Hi Beate,
I have successfully purified membrane proteins from IBAs Strep-Tactin (superflow, high capacity) with either 0.03% DDM or 0.25% DM. My Strep-Tag is also at the C-terminus. Strep-Tags like to insert into existing secondary structures in crystals (we had a beautiful example in a soluble TIM-barrel protein, where the Strep-Tag inserted into an extended beta-sheet of the neighboring molecule, but in solution the tag was accessible). I guess you solubilize with 1% DDM/DM? I do that for 1 hour, then centrifuge (50.000 rpm, ultracentrifuge for 1h) and then dilute the supernatant to a final detergent concentration of 0.2%. You could try to apply your protein in batch manner on the Strep-Tactin resin for 1-2 hours while rotating. I use only 20 mM Tris - 100 mM seems a bit too much for me (Tris is an amine. which could reduce and harm your resin/protein). Good luck,
Magdalena
It does sound like that if only 50% sticks that you have overloaded the column. It is difficult from your description to tell what had happened.
If 50% sticks, then after you wash and elute does anything come off? Or is your protein only detected in the flow through, ie the material that doesn't stick?
If nothing comes off, then it is likely because you changed the detergent concentration and/or left out the glycerol and maybe your protein precipitated under these conditions.
@Magdalena Schacherl - 100 mM Tris is the recommended buffer for Strep-Tactin column
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