Limitless suggestions, but as you are about to embark on TEV digestion, one thing you can do is do this within a dialysis tube and 1:20 and dialyse for the magic period (overnight) to get rid of the imidazole, then follow this with an IMAC step to remove uncleaved protein and the TEV. Remember to equilibrate your IMAC column including a little imidazole 5-30mM because, despite your wanting your tagged stuff to stick, some proteins stick to the resin anyway if you don't equilibrate. To make doubly sure of this I always elute off the beads and run it on a gel, just to see what is going on.
cheers charlie > 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. > -- Dr Charles Allerston Associate Research Scientist Structural Biology Ark Therapeutics Darwin Building, UCL Gower Street, London WC1H 9EH United Kingdom mailto: c.allers...@ucl.ac.uk phone: +44 (0)2076791354 Web: www.arktherapeutics.com
