I have always performed TEV cleavage after eluting my protein from the Ni-NTA column (TEV cleavage performed during overnight dialysis in imidazole free buffer as imidazole inhibits TEV activity at high concentrations). I use TCEP as reducing agent and have never included EDTA, by this method I've never had a problem with membrane or soluble proteins and get almost 100 % cleavage of the tag.
Best, Jon On Tue, 31 Oct 2023 at 19:21, 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/
