Hi Ronaldo, We immediately desalt after His-Trap (desalting column connected in series) into an Imidazol free and low salt buffer and digest with 1:50 to 1:100 TEV:substrate ratios (w:w) at RT or in the cold room (time often a compromise between protein precipitation and completion of the cut) before proceeding to ion exchange and size exclusion chromatography.
cheers, Ralf -----Original Message----- From: CCP4 bulletin board on behalf of na...@icb.ufmg.br Sent: Wed 9/9/2009 8:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off topic: Purification steps 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.
