Hi

I am stuck this purification step since last one year

my periplasmic protein ccNiR have leading sequence at N terminal, so I added 10 His tag at C terminal. I used FPLC and Ni column to purify my protein. But when I tried to wash off my protein by increasing Imidazol 250mM pH 8.1 gradient, I always get a smear band in my FPLC purification at 280nm instead a sharp band at fixed Imidazol percentage ( concentration). My protein is not coming out at a fixed concentration. Is His tag digested by carboxypeptidase enzyme (Swanella Bacteria) or any other reason for this smear band? How can I purify my protein at a fixed Imidazol concentration ?

Thanks in advance.