Re: [ccp4bb] Off topic: Purification steps
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
Re: [ccp4bb] Off topic: Purification steps
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.
Re: [ccp4bb] Off topic: Purification steps
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
