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 LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China Date: Mon, 26 Mar 2012 15:04:43 +0300 From: peg...@pasteur.gr Subject: [ccp4bb] recommendations_on_purification To: CCP4BB@JISCMAIL.AC.UK 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
