There is non specific elution of multiple proteins in which keep on reducing with increasing concentration of imidazol , along with target protein. I routinely use 500mM NaCl in samples and wash 1 of 20mM Imidazol and Wash 2 of 50mM Imidazol. What can I do to remove non-specific elution?
Ellis C O'Neill
Try eluting with a gradient - non specific proteins should elute first and your protein of interest hold on until higher imidazole.
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Soumya Mohanty
Try using cobalt based resin such as Talon from Clontech instead of Ni-NTA. In my experience, the non specific impurities were reduced significantly. There might be a reduction in the overall yield of your target protein though as cobalt is much more specific to polyhistidine tag.
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