Phoebe, It just so happens that we've been working on a C4 Zn-finger (or something very much like a Zn-finger) very recently without knowing that we had one (it's in the PheRS from S. haemolyticus, link to paper below) and we've purified the protein w/o any issues on an IMAC column (His-SELECT). When we found the metal site in the structure, we sent the protein out for metal analysis (since we did not know for sure what metal(s) occupy the site) and the Zn was all there within 10% error margin (based on expected molar ratio of 1:1, protein quantity by necessity estimated from protein concentration), so no exchange with Ni or other metals took place.
This follows the trend I noticed with other C3 and C4 Zn-fingers - they generally seem to bind Zn so tightly that no appreciable exchange takes place under 'normal' conditions. C2H2 may be different - I have no direct experience with that sub-class. Again, only based on my personal experience (literature cases probably exist that indicate otherwise), Zn(CX)4 chelate does not seem to bind to Ni-NTA, His-Select, or Talon resin. Incomplete assemblies (i.e. Zn-free) may bind which is why it's probably a good idea to add a tiny amount of Zn into the lysis buffer, to make sure that all the incompletely chelated species are 'filled in'. Again, based on personal experience - these 'empty' species tend to form nasty aggregates which fall out of solution ASAP, anyway. Please note that the above is applicable to Zn fingers that supply all four ligands from the same protein chain. When two chains supply the ligands, many more nasty things become possible. Hope this helps, Artem P.S. the paper is linked to http://www.xtals.org/pubs.html, first link in the Anti-bacterial section. -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Thursday, January 03, 2008 7:21 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Zn fingers and Ni columns This has probably been discussed before, so apologies in advance. We're eyeing a protein that has a probable C4 Zn finger in the middle. The collaborators who are nicely going to PCR it up want to know if we'd like it with or without a His tag. Is it a bad idea to co-mingle Zn-binders and Ni columns? Or is it likely to bind the column quite nicely without the tag? thanks, Phoebe ---------------------------------------------------------------------------- ----------------------------------------------- Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 fax 773 702 0439 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alpha betically.php?faculty_id=123 http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
