Dear Rafael In addition to the excellent suggestions already offered by previous responders, I can attest that Ni-penta resin (sold among others by a company with an odd name Marvelgent) is resistant to EDTA and DTT, and it leaks very little Ni (almost none). Plus, it elutes with low inidazole, owing to the single available chelation point offered by the immobilized Ni ions.
Depending on the resin and other conditions tou use, there generally is no reason to worry about the small amount of DTT and EDTA that may be present in TEV preparations. On the other hand, there is a reason to be slightly concerned that the leaky Ni ions may inhibit your TEV. In summary, you have to try it on small scale, the odds are good that cleavage on resin will work for you. Notably it even works when TEV itself is his-tagged, presumably owing ro the dynamic nature of His/Ni interactions (strictly speaking, resin affinity is only about 1 uM, but avidity and intra resin exchange and recapture keep the bulk of protein trapped in the resin). Good luck! Artem On Tue, Oct 31, 2023, 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/
