Properly made TEV protease should work with or without rocking. We have done QC of our TurboTEV, a dual GST- and His-tagged TEV, under many conditions and used on hundreds of proteins. The most common cause of incomplete digestion is the insolubility of target protein or bad construct design. Shaking or agitation may oxidize the protein. TEV is a Cys protease that can be oxidized as well. Adding reducing agents or EDTA may well prevent some of the effects of agitation. Unlike plasmid DNA, protein solutions should be mixed GENTLY. Regarding the original question, 50% loss may not be a bad thing if it is about total protein. In our experience, it is not uncommn to have only 50% recovery after tag removal of a Ni pool. Some proteins aggregate after tag removal even it's His-tag. Again the loss can be a good thing. Chun Accelagen -----Original Message----- From: Laurie Betts <laurie.betts0...@gmail.com> Sender: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> Date: Thu, 31 Mar 2011 11:11:33 To: <CCP4BB@JISCMAIL.AC.UK> Reply-To: Laurie Betts <laurie.betts0...@gmail.com> Subject: Re: [ccp4bb] Question about TEV cleavage
We have the same experience with the DO NOT AGITATE. We purify our own His-tagged TEV protease, and flash-freeze it IMMEDIATELY after IMAC prep at about 1 mg/mL. Laurie Betts UNC On Thu, Mar 31, 2011 at 10:37 AM, Charles Allerston < charles.allers...@sgc.ox.ac.uk> wrote: > I echo this. > > Some time ago I was working on a target which seemed to precipitate when > cleaving overnight with TEV. I wasted a fair bit of time trying to optimise > cleavage conditions with a myriad of buffers. In the end, just by not > agitating my solution, there was no precipitation and recovery was extremely > good. > > I never agitate my solutions when cleaving now and have not had any > problems with cleavage (to do with this issue, anyway) or recovery. > > > cheers > charlie > > > > Dr. Charles Allerston > Genome Integrity Group > Structural Genomics Consortium > Nuffield Department of Medicine > Old Road Campus > University of Oxford > OX3 7DQ > http://www.sgc.ox.ac.uk/ > > > > > > -----Original Message----- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Xiaopeng Hu > Sent: 31 March 2011 15:23 > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Question about TEV cleavage > > Our experience is do not shake the tube during TEV cleavage,I dont know > why, but it does help. > > xiaopeng >
