Hi Rafael, the simple answer is that the EDTA and DTT( or any other reducing agent) is not required for the TEV activity. You can have your TEV protease in 20 mM Tris pH 7.5 (RT) and 150 mM NaCl, plus either 10% Glycerol or 50% glycerol depending on storage conditions preference - 80 or -20C respectively.
Normally you need to take care that you are dilating the TEV several fold (at least 5) in order to drop the Glycerol concentration below 10% not to have an inhibitory effect on the activity. Also your on-column cleavage buffer should not contain higher than 300 mM salt, otherwise the TEV activity will be decreased as well. Go ahead and give it a try. Good luck! Kind regards, Nikolay On Tue, Oct 31, 2023 at 3:21 PM Rafael Marques <rafael_mmsi...@hotmail.com> wrote: > > Hi everyone, > > I have been looking on this bb and other websites as well but I could not > find a veredict. We are suspecting that when I elute my sample from my Ni-NTA > column, the imidazole concentration (250 mM) is making it to precipitate. > Once my sample has a cleavable TEV site, I was planning to incubate my loaded > resin overnight with TEV and get my sample back simply using my lysis buffer. > And here lies the problem. Most of the TEVs are kept in EDTA and DTT and I > wonder if they are essential for its protease activity or if I could use > another reducing agent more compatible with my resin (or maybe do not add > both). I saw that someone did not have EDTA and used b-mercap. instead of > DTT. May I have your comments if you guys already faced a similar situation? > > Best wishes > > ______________________________________________________ > > Rafael Marques da Silva > > PhD Student – Structural Biology > > University of Leicester > > > Mestrando em Física Biomolecular > Universidade de São Paulo > > Bacharel em Ciências Biológicas > Universidade Federal de São Carlos > > phone: +55 16 99766-0021 > > "A sorte acompanha uma mente bem treinada" > ________________________________________________ > > ________________________________ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
