Katherine, You are not alone. I have inadvertently destroyed a GE HisTrap column with high concentrations of proteins that contain many exposed cysteines. In my case the Co2+ resin turned a very dark purplish-brown and the protein appeared to have crashed out on the column. I didn't try to strip it, because I figured it was done for anyway, so I can't tell you any more about the problem. Here's how I explained it to myself (whether or not this is actually right I'm not 100% sure, but it makes sense in my head). The columns I was using have a maximum concentration of 5mM for DTT and 10mM for B-mercaptoethanol. So that seems like the column can handle 10mM thiol groups. If you have a protein with many cysteines and it is very highly concentrated (as was the case for me) then you are adding considerably more thiol groups to the solution. This abundance of thiols reduces the metal on the column, and disaster ensues. For me, repeating the same prep with less DTT (3mM vs. 5mM) in the buffer fixed the issue. If you are concerned about your protein oxidizing at lower concentrations of DTT or BME, the other alternative is to switch to TCEP. The IMAC columns can tolerate higher concentrations of TCEP, and it is a far superior reducing agent (more stable, more reductive, etc.)...but also a lot more expensive (although you can get away with using much less because it works so much better).
HTH, Mike ----- Original Message ----- From: "Katherine Sippel" <katherine.sip...@gmail.com> To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, January 12, 2012 4:01:10 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Metal won't strip from IMAC 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 -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
