What is the maximum concentration of imidazole that can be used to elute strongly bound protein from Ni-NTA column?

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Francisco Solano

If 0.5 M is not enough, try to change pH. To increase above 0.5 M is not effective

Heide Marie Resch

What buffer condition are you currently using? You could for example increase the NaCl concentration in case your protein is sticking unspecifically to the column matrix.

Stefan Gajewski

2 M imidazole in 500 mm NaCl should be fine. Make sure to adjust the pH of your final buffer.