90% of the protein could be aggregated and hiding the His tag from the resin. Metal ions might be removed from the starting sample by adding up to 10 mM EDTA in the first buffer exchange cycle, but just after reading "10% of the protein binds" I wouldn't bet much on this. Rather increase the culture volume and move on.
Carlos Kikuti Em 26/03/2012, às 14:39, Lorenzo Finci escreveu: > Petros, > > It has indeed been speculated that high concentrations of Magnesium and/or > other metals present in the cell lysate effect the binding of the > Histidine-tag, and thus specific conditions for binding and elution need to > be optimized for specific elution of your target protein. I believe that the > standard recommendations when using a Nickel column to bind to your > Histidine-tag is that you can try using 10-20 mM Imidazole to wash unwanted > unspecific binding protein, to try manipulating the binding by lowering the > pH of the buffers, and by determining the specific concentration of . > Alternatively, you can also try using another metal column such as Cobalt > (Talon) with a higher affinity for the Histidine tag. > Sincerely, > lorenzo > > > Lorenzo Ihsan FInci, Ph.D. > Postdoctoral Scientist, Wang Laboratory > Harvard Medical School > Dana-Farber Cancer Institute > Boston, MA > Peking University > The College of Life Sciences > Beijing, China > > > > Date: Mon, 26 Mar 2012 15:04:43 +0300 > From: [email protected] > Subject: [ccp4bb] recommendations_on_purification > To: [email protected] > > 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
