Are you treating all your buffers with metal chelating resin? Are you washing all plasticware with EDTA and metal-free buffer prior to use? How are you quantifying your metal content, and what metal ions are contaminating your sample? You might be pulling out the metal ions, but they get right back in as soon as you remove the chelators. Metal ions are present at trace levels in all water and chemicals (Zn is particularly common). You might need to 'soften up' the protein a little bit, we use a pH 3.8 stripping protocol. Denaturants may be required. Time is also a factor, extending your chelating step might help. It's not trivial to make app-enzymes, in my experience, Nat
On Mon, May 19, 2014 at 12:20 PM, SUBSCRIBE CCP4BB Adam Brummett < adam-brumm...@uiowa.edu> wrote: > Hello All, > > I apologize for the non-crystal related question. I am trying to get a > fully metal-free apo enzyme. The 6x His construct is consistently purified > with some metal (20-30%). I have attempted chelating away the metal with up > to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little > to no effect. Any thoughts or recommendations would be greatly appreciated. > Thanks. > > Adam >
