Hi Katherine, I recommend Zn-IDA Sepharose (Chelating Sepharose Fast Flow, GE Healthcare), which we have been using successfully for more than 20 years, since the early days of IMAC:
Skerra et al. (1991) The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a generally applicable purification technique. (Nature) Biotechnology 9, 273-8. This metal/chelate combination has exquisite selectivity for the His6-tag, at least if operated with an imidazole concentration gradient. Importantly, Zn(II) typically forms reversible sulfide complexes and it is not redox-active (in contrast with Co, Cu, Ni)! In deviation of our old protocol I would just recommend to use a concentrated ZnSO4 stock solution (instead of ZnCl2), which is less prone to hydrolysis upon longer storage. Good luck, Arne Am 13.01.2012 um 01:01 schrieb Katherine Sippel: > Hi all, > > I've run into a bit of a protein purification conundrum and wondered if > anyone had encountered a similar situation. I've exercised all of my > google-fu and can't find anything. It's a fairly straightforward setup; > His-tagged protein and Talon Co2+ resin, load lysate, wash with 5 mM > imidazole, elute with 150 mM imidazole. There is protein in the elution > fractions as would be expected. The strangeness occurs when I try to > regenerate the column. Using the standard protocol of 25 mM MES, 100 mM NaCl > pH 5 doesn't change the color of the resin back to light pink the way it > should with a regenerated column. I try stripping with the suggested 0.2M > EDTA, still pink, 0.5M EDTA, still pink, 8 M urea plus 4% CHAPS and then > EDTA, still pink, 1 M NaOH then EDTA, still pink. I've checked the resin > using a Western (with a really specific monoclonal Ab) and it seems that my > protein has somehow irreversibly bound to the column and is preventing the > metal from releasing the sepharose. I've even tried competing the protein off > with excess Co2+ and Mg2+ (the endogenous divalent bound cation). > > Clearly the solution is swapping to a Ni column, but this is really bugging > me now. Has anyone run into this problem with IMAC before? > > Background: The protein does bind divalent cations (Mg and Mn) with low > affinity (~1 mM) and has a ridiculous number of cysteines (10 in 416 residues > total). There is 1 mM BME and 1 mM MgCl2 in all of the buffers. > > Thanks, > > Katherine + Prof. Dr. Arne Skerra + Lehrstuhl f. Biologische Chemie + Technische Universitaet Muenchen Emil-Erlenmeyer-Forum 5 + 85350 Freising-Weihenstephan + Germany Phone: +49 (0)8161 71-4351 + Fax: -4352 http://www.wzw.tum.de/bc + eMail: ske...@tum.de
