Discoloration of Nickel affinity resins during 6-his protein purification - (Oct/21/2013 )

For the last year I have been plagued with a problem when I purified my 6-his labeled protein-- my resin would become a reddish-brown color that could not be washed away. The color was eluted off when I eluted my protein and I had problems separating it from my protein. I was isolating material from mammalian cell tissue culture.

I am an idiot. I was co-purifying transferrin, an iron binding protein that is prevalent in tissue culture supernatant, and will also bind nickel. At some point I stopped loading my material in the presence of 20mM Imidazole. When I re-initiated this practice any color was much less, and the column could be reused upon washing.

I would read posts about this problem on this site, which is why I am posting this advice. You must load your starting material in the presence of a low concentration of Imidazole. Especially if you are purifying proteins from tissue culture supernatant.

Thank you very much. I think this is very usefull for me. I will purify protein from mammalian cell some day.