Re: [ccp4bb] Question about TEV cleavage

2011-03-31 Thread Peter Hsu
We've been using the S219V mutant for cleaving out tags. We usually do our cleavage reactions in an overnight dialysis after a Ni-column into 50mM Tris, 100-200 mM NaCl, 5 mM BME at 4C. I've never found problems with losing any protein in the reaction and recover usually >90% of the protein afte

Re: [ccp4bb] Question about TEV cleavage

2011-03-31 Thread Zhijie Li
Chun Accelagen From: Laurie Betts Sender: CCP4 bulletin board Date: Thu, 31 Mar 2011 11:11:33 -0400 To: ReplyTo: Laurie Betts Subject: Re: [ccp4bb] Question about TEV cleavage We have the same experience with the DO NOT AGITATE. We purify our own

Re: [ccp4bb] Question about TEV cleavage

2011-03-31 Thread Chun Luo
aurie Betts Sender: CCP4 bulletin board Date: Thu, 31 Mar 2011 11:11:33 To: Reply-To: Laurie Betts 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

Re: [ccp4bb] Question about TEV cleavage

2011-03-31 Thread Klaus Piontek
And not at full moon! Klaus Am 31.03.2011 um 16:23 schrieb Xiaopeng Hu: Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H

Re: [ccp4bb] Question about TEV cleavage

2011-03-31 Thread Laurie Betts
; 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 > Xiaop

Re: [ccp4bb] Question about TEV cleavage

2011-03-31 Thread Charles Allerston
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

Re: [ccp4bb] Question about TEV cleavage

2011-03-31 Thread Xiaopeng Hu
Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng

[ccp4bb] Question about TEV cleavage

2011-03-31 Thread Prince, D Bryan
Hello all: I am curious about what level of recovery is reasonable when performing a TEV cleavage to remove 6HIS tags (N-terminal) from a protein. Currently we are experiencing >50% loss of soluble protein after TEV cleavage, and feel that this is too much. Are there better systems for his tag cle