I suspect that sometimes the protein chaperones the tag, which is solvent exposed some fraction of the time. Try very slow loading or batch binding.
Kendall Nettles On Mar 26, 2012, at 8:15 AM, "Petros Giastas" <peg...@pasteur.gr<mailto:peg...@pasteur.gr>> wrote: Dear all, I am expressing a 6xHis tagged secreted protein in a fermentor in P. pastoris, using the standard minimal medium described in the invitrogen manual (plus PTM1). Following collection of the culture medium, I am having problems with purification of the protein as only a small fraction (~10%) binds to the Ni-NTA beads even after extensive buffer exchange (when expressed in full BMGY media this is not observed). Could this be attributed to metal ions still present in my sample? Is it likely to be due to poor protein quality in this medium? Or any other suggestions? Thanks in advance Petros
