Imidazole can also be used across a gradient to obtain purer protein. Proteins will be more stable and more active at the neutral pH of imidazole purification (unless inhibited by imidazole). Proteins are generally less stable and less active at low pH. Those that are stable may not be stable at the starting pH (8.0). I would recommend using imidazole - make sure you use a high purity supply which does not absorb at 280 nm for monitoring by UV absorption.

As OP says, if you elute with pH and you aren't completly sure what you're doing, the protein may denaturate or precipitate inside the column, since you may reach the isoelectric point (not only for your protein, but for others that interact with the matrix aswell). From my experience it's better to do it with imidazole. If you have some way to track your protein, I would do a gradient cromatography at first, searching at which imidazole concentration does your protein elute (consider this as an aproximation, you should check higher and lower concentrations). Then you can do the procedure in cromatographic steps with different imidazole concentration.

I would elute with imidazol-buffer with a pH-value of 7.0 Than you combine both ;-) In my hands this works very well.

Proteins are generally less stable and less active at low or very high pH. Elution of protein using pH gradient requires a considerable amount of lowering of pH (as low as pH 4-5) which may results in protein denaturation.

The Ni-NTA columns are designed for purification of 6xHis-tagged recombinant proteins expressed in bacteria, insect, and mammalian cells. The general method is to absorb the protein onto the column, by mixing the beads with the sample, then pouring the slurry of NTA beads and protein into a column, where low concentrations of phosphate and imidazole are used to remove low affinity bound proteins. If needed, the imdazole can be increased to 20 mM before most His tagged proteins are eluted. Finally, higher concentrations of imidazole is used to elute the protein from the NTA-beads.

For proteins sensitive to low pH, competitive elution with imidazole at nearly neutral pH or even at pH 8.0 is more favorable. Imidazole is included in the Native Wash and Elution buffers to minimize the binding of untagged, contaminating proteins and increase the purity of the target protein with fewer wash steps. Note that, if level of contaminating proteins is high, imidazole can also be added to the Native Binding Buffer.

Native binding buffer: 10 mM of imidazole, pH 8.0

Native wash buffer: 20 mM of imidazole, pH 8.0

Native elution buffer: 250 mM of imidazole, pH 8.0.

Thank you very much for all the comments! It really helps. I will rather use imidazole for native purification.

The choice of an elution protocol depends a lot on protein stability. If you prefer a low pH elution you could incubate your column with a low pH buffer while performing a flash elution. The moment you collect your protein fraction you could readjust your elution pH to 7.5 or 8.0. This may help in maintaining your protein's stability. In a high urea buffer imidazole appears less effective to elute, however in my experience it has worked for elution after on-column refolding only.