I have a his tagged protein which I am trying to purify by Ni-NTA column. The buffer that I am using for purification is phosphate buffer saline (di sodium hydrogen phosphate and sodium di hydrogen phosphate). I have used 10mM-50mM imidazole for washing. After 50mM washing no band of protein of interest was observed at 250mM elution whereas if I do washing with 40mM imidazole , there is elution of other non specific protein with protein of interest. I have also tried eluting my protein at 150mM imidazole but still the non specific protein were seen on PAGE. Probable size of non specific protein are near by 65kda and 50kda . please let me know how can i minimize the elution of these non specific protein

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