Hi Rafael, For completeness - there are alternatives to imidazole for eluting proteins from Ni-NTA resins:
EDTA - (strips Ni2+, and therefore everything bound to the Ni2+) - 50mM should be sufficient, in your favourite buffer/salt system. Obviously not appropriate for metalloproteins. You'll need to recharge the column/resin afterwards. Low pH - Citrate or Acetate buffer with a pH lower than 5.5 (lower still for multimers) with an appropriate salt concentration. Obviously test this on a small scale first to see if your protein tolerates the drop in pH. You can reduce the time of exposure to low pH by eluting the protein straight into some 1M Tris or HEPES to bring the pH back up to something more neutral. Hth, Dave Dr David C. Briggs CSci MRSB Principal Laboratory Research Scientist Signalling and Structural Biology Lab The Francis Crick Institute London, UK == about.me/david_briggs<http://about.me/david_briggs> ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Rafael Marques <rafael_mmsi...@hotmail.com> Sent: Tuesday, October 31, 2023 7:21:21 PM To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage External Sender: Use caution. 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 The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT ######################################################################## 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/
