I am trying to purify a protein with pI 6.4. I use Tris buffer at pH 8, 500mM NaCl for the purification. For homologs of the protein, I have a standardized protocol, where I use urea during lysis, but not for purification from Ni beads and ion exchange (refolding of the protein is not a problem).
This protein however, precipitated on Ni beads, and I had to use denaturing buffers with 6-8M urea to elute it. Reducing the concentration of urea below 6M also precipitates the protein. I tried using 10% glycerol, but that also precipitates the protein. I can use urea throughout the purification process, for Ni affinity, and Mono Q column. However, the problem is I need to use it for ITC finally, and I cannot use urea for that, because the interacting protein would unfold.
How big is this protein? Does it need metals or cofactors to fold? Do you expect it to be folded, that is, have you checked a secondary structure prediction? Or do you expect the protein to fold upon binding to its partner? Sometimes expressing the protein as a fusion can help keep it in solution. Tags like MBP can be useful for larger proteins and often don't interfere with affinity assays.
Try Buffers at pH >8 upto 10 sometimes you have to go a few more points above pI than you think.
Addition of proline or arginine to the buffer at upto 0.4 M solubilises protein by reducing charge interactions.
Low levels of detergent e.g. 0.05% Tween20 can stabilise protein in solution.
Reduce the salt concentration - try a range from 150 mM
If there are free cysteines, add DTT or TCEP at 1 mM, this can stop aggregation.
I hope this helps. Do let us know how it goes. Good luck!
Have you tried lowering the NaCl concentration? NaCl can be chaotropic and the high ionic strength can also enhance hydrophobic interactions that could lead to protein aggregation.
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