Hello all,
I am currently trying to purify a fragment of the large surface protein of HBV however during purification I notice that a white precipitate form on the Ni-NTA column. (At pH 8.0; pI of protein = 6.97) I'm using a simple lysis buffer (50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, (5% glycerol in some cases but no significant difference))
The protein was in solution in the cell lysate, ie before loading onto the column and the precipitate is confirmed to be my protein.
Washing and eluting at pH 8 resulted in a milky, cloudy white eluate.
Washing and eluting at lower pH of below 6 has helped to solubilize protein and the solution is clear.
For subsequent experiments I need to bind my protein to Ni-NTA carrying liposomes but as soon as my protein comes into contact with them it precipitates. The protein also precipitated when adding a bit of NiSO4 solution.
Any ideas as to how I can avoid this issue?
Hi,
It seems to me that it's the Ni ions that are causing the problem. perhaps when they bind to the his tag they cause the protein to destabilize. You could try using a different affinity tag system and see if that fixes the problem. Biotin/avidin is probably where I would start. If you want to stick with the his tag, you could try moving it to the opposite terminus.
Why do you not prefer to conduct the purification at pH 6? Should you strictly perform at alkaline pH?
Did your protein also precipitate when adding a bit of NiSO4 solution at both pH 6 and 8?
Do you observe the same precipitation when using IMAC columns with immobilized Cobalt, Copper or Zinc ions? You can basically screen all the first row transition metals for that matter.
Good luck!
For Ni-NTA columns, pH of wash and buffers should be kept in the acidic range. You said washing and eluting at 6 ph helps clarify the solution. Try ph around 5-6. Hope this helps.
Reference: Bornhorst, J. A., & Falke, J. J. (2000). [16] Purification of proteins using polyhistidine affinity tags. In Methods in enzymology (Vol. 326, pp. 245-254). Academic Press.
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