If the protein is bound so tightly to the nickel immobilized on the beads that it won't come off even with 1M imidazole, there are a few possibilities. If the protein has a very high affinity for Ni on account of having a His12 tag, for example, you could elute the nickel from the beads using EDTA, causing the protein to elute also, then dialyze out the EDTA-Ni complex. A change in pH might help as well.

Another possibility is that the protein is stuck on the column due to having precipitated there. This could happen to a soluble protein if the pH is very close to the pI of the protein. Changing the pH might help.

A mild non-ionic detergent would help keep the protein in solution if it is not soluble on account of being hydrophobic. I don't think it would help elute a soluble protein from Ni beads if the protein has a very high affinity for the Ni. These detergents are hard to remove, if that is needed, but on the other hand, you wouldn't want to remove them if they were keeping the protein in solution. They may not be a problem for the analyses you plan.

If a specific detergent is a problem (for example the fluorescence or UV absorbance of Triton X-100), then choose a different one. If you expect to need to remove the detergent, use one that is easily removed by dialysis, such as CHAPS.

If the protein is bound so tightly to the nickel immobilized on the beads that it won't come off even with 1M imidazole, there are a few possibilities. If the protein has a very high affinity for Ni on account of having a His12 tag, for example, you could elute the nickel from the beads using EDTA, causing the protein to elute also, then dialyze out the EDTA-Ni complex. A change in pH might help as well.

Another possibility is that the protein is stuck on the column due to having precipitated there. This could happen to a soluble protein if the pH is very close to the pI of the protein. Changing the pH might help.

A mild non-ionic detergent would help keep the protein in solution if it is not soluble on account of being hydrophobic. I don't think it would help elute a soluble protein from Ni beads if the protein has a very high affinity for the Ni. These detergents are hard to remove, if that is needed, but on the other hand, you wouldn't want to remove them if they were keeping the protein in solution. They may not be a problem for the analyses you plan.

If a specific detergent is a problem (for example the fluorescence or UV absorbance of Triton X-100), then choose a different one. If you expect to need to remove the detergent, use one that is easily removed by dialysis, such as CHAPS.

Thank you Adam, I should give a try with EDTA and playing with pH.

Hi Amit

Adam had some very helpful suggestions.  May I extend upon these:

 - Have you had enough protein to perform an IEF to determine pI?

-  I have looked up a predicted pI using Swiss Prot back in the day - that helped me make decisions upon operating pH of my purification system. 

- The previous step helped me boost yield to then confirmed the functional pI ranges by IEF. 

- Stick with Dialyzable detergents (Adam is correct), I have used glycerol up to & not exceeding 5%.  Glycerol will work if your product needs to be only immunoreactive.  If you need to perform X-ray, nmr analysis, the glycerol may get in your way and the Triton & Tween skeletons may as well. 

I would be interested in learning of your downstream usage for your recombinant protein.

The recombinants I produced were used downstream for immunodiagnostic device development - fun devices to build and manufacture. 

Best Regards