Dear Ronaldo! I think this should be very straight forward, Generally, after Ni-column protein can be digested with TEV (1:50, or 1:100 ratio) either at room temperature or at 4C depending on the stability of the protein. I had tried this at room temperature, overnight cleavage at 1:100 (TEV: Substrate) ratio. After TEV digest you can load it onto the His-trap column again by reducing the concentration of Imidazole by half or 1/3rd of the original concentration at which protein was eluted. TEV digested protein will remain unbound to His-trap column and will be in flow through. TEV with his tag will remain bound to Ni-column. Finally Imidazole can be removed during the concentration process by buffer exchange.
Raj On Wed, Sep 9, 2009 at 8:48 AM, <na...@icb.ufmg.br> wrote: > Dear CCP4bb users (this is an off-topic question), > > We have a few proteins being expressed as > HIS-TAG_(TEV_cleavage_site)_PROTEIN > and we are about to initiate the purification steps. > > We have already used the HiTrap-Chelating columns from GE for the first > purification step (affinity chromatography) and we would like to move > forward > with TEV digestion and a second purification step. > > We know these following steps are very protein-dependent, but we were > wondering > one could share his/her experience in the following steps: removal of > imidazole, > cleavage protocol and cleavage identification, second chromatography, etc. > > Any experience would be appreciated. > > Thanks in advance > > Ronaldo. > -- Rajendra Kumar Singh
