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: [log in to unmask]
|