I can only say that I've purified dozens of metalloproteins using IMAC and have so far not noticed many issues with metal ion losses. Having said this, I also must add that I've been exclusively using His-SELECT resin (unintentional but well-deserved plug for Sigma) for the past few years, and therefore the concentration of imidazole used for elution was consistently less than 100 mM. The same may be accomplished using e.g. Co-Talon resin, but unlike Talon the protein-binding capacity of His-SELECT is quite high.
It is possible and even likely that for some proteins imidazole is going to be an issue. In fact, we've had exactly such a case relatively recently - where the protein had to be passed from Ni-NTA column directly onto a desalting column in order to minimize exposure to imidazole. The problem was ultimately avoided by switching to His-SELECT and lower imidazole elution. Artem _____ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jacob Keller Sent: Thursday, July 17, 2008 8:04 PM To: [email protected] Subject: [ccp4bb] Imidazole's ability to chelate metal ions Dear Crystallographers, Does anybody happen to know whether imidazole is able to chelate metal ions in solution? It seems reasonable that since it can compete for binding to IMAC resins, it should have some affinity for at least Ni++ and Co++, but what about metal ions like Ca++ and Mg++? I assume that the affinity is weak, but at the concentrations at which we are wont to use it in our elutions (~100-500 mM), does it not seem likely that other metal ions are being competed away from our proteins as well? Jacob Keller ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *******************************************
