Hi Rafael, Dom et al, Indeed nickel eluted with imidazole stays on all your proteins with a His tag, and addition of DTT will make almost all proteins go brown and crap out at this point. But, as Dom says, addition of EDTA before the DTT will solve the problem. Most of the time…
If your protein goes brown after a nickel column and you thought it was something special to your protein, try again..! If your protein can’t handle EDTA, try the pH trick suggested in another post. If none of that works, then move to GST or some such… Ed https://www.ularkin.org/team-member/thomas-edwards/ Sent from my iPhone. On the run… On 31 Oct 2023, at 16:48, Dom Bellini <dbell...@mrc-lmb.cam.ac.uk> wrote: Hi Rafael, Once I inherited a protocol with a problem similar to yours and they told me that the precipitation was caused by nickel leaking out during the elution with 250 mM imidazole. I am not sure whether this was true, however, their fix was to place something like 20 ul of 200 mM EDTA at the bottom of the collection tubes into which the protein was eluting. This indeed kept the protein soluble, at least long enough to dialyse or gel filtration. Good luck! D On 31 Oct 2023, at 19:21, Rafael Marques <rafael_mmsi...@hotmail.com> wrote: CAUTION: This email originated from outside of the LMB. Do not click links or open attachments unless you recognize the sender and know the content is safe. .-owner-ccp...@jiscmail.ac.uk-. 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 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/
