Hi, In my hands, I have found the addition of TCEP (~0.5 mM) helps with TEV cleavage efficiency, especially reactions at 4C where TEV is much less efficient anyway. The caveat is that my targets may have been "happier" with TCEP present, therefore encouraging more efficient cleavage. The variance in what you've been able to find online, and reflected in the responses thus far, could be down to different labs using different TEV constructs. Older constructs seem to be more sensitive to reducing agent presence whereas optimised versions of TEV are much more efficient so any drop off is less noticeable. It may be worth looking at your cleavage buffer composition too - not too much salt, glycerol, detergents etc
As for on-column TEV cleavage, if your TEV is also His-tagged then it will likely be less efficient. If you're willing to try a new construct, I have had great success with on-column cleavage using target bound to Streptactin XT resin. If you want to stick to Ni-based IMAC, Cytiva's Ni Excel, BioRad's Profinity and Protein Ark's Fastback Ni Advance all leach less than conventional Ni-NTA/IDA under less than ideal buffer conditions. Good luck! Irwin ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Hughes, Jonathan <jon.hug...@bot3.bio.uni-giessen.de> Sent: 02 November 2023 09:17:30 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] AW: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage (this is from a different jon) if a chelator really is necessary at this stage (post AmS?), in any case use IDA instead of EDTA! that's pretty obvious from the affinities of EDTA/NTA/IDA. cheers jon Von: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> Im Auftrag von Jonathan Bailey Gesendet: Mittwoch, 1. November 2023 19:53 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage 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<mailto: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 ________________________________ 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/
