This is a fairly familiar issue. A hydrophobic protein with thiols often contaminates IMAC columns and this kind of contamination can be difficult to remove completely. In the past some of the methods that worked included trypsin or papain treatment, followed by massive amounts of chelating agents. I would like to note, however, that the cost of such 'rejuvenation therapy' easily approaches the cost of 5-ml resin batch. In general with IMAC it is almost always easier to perform the first step in batch, since it takes less time for larger samples. So unless your column is very large and expensive it sounds cheaper to get a new bit of resin...

On Thu, Jan 12, 2012 at 6:01 PM, Katherine Sippel <[log in to unmask]> wrote:

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