Hello Matthew, I almost always charge my IMAC resin with Co instead of Ni. I find that it gives much better purity because the selectivity is better. This is because the coordination sphere around the Co atoms require more His residues than the Ni coordination sphere. I believe Co requires 4 His, while Ni only requires 2. That being said, although it is more selective, proteins with a His tag are likely to have lower affinity for Co, and will therefore elute in lower imidazole concentrations, and if your His tag is not entirely exposed, you may have issues getting it to bind at all. I have never had this problem, but I have heard that it can be an issue. It is probably worthwhile to try cobalt, but do it under the assumption that there is a small chance you will have to go back to Ni.
Also, if you use cobalt, you may notice a slight color change in the column when you run it under certain buffer conditions. Some additives cause the color to change from light pink to a purplish color. This is ok, the column will still bind your protein fine. Just be sure to look in the resin manufacturer's manual to make sure you don't have any incompatible components in your buffer. In general, anything that can go over the column when it is charged with Ni should be ok for Co as well. Good luck, Mike Thompson ----- Original Message ----- From: "Matthew Bratkowski" <mab...@cornell.edu> To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, July 22, 2010 7:05:21 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Quality of His-Select Resin After Regeneration Hi. We have been using His-Select Resin from Sigma in our lab for a number of years now. When we first bought the resin, I usually got much better purity of His tagged proteins compared with regular Ni-NTA resin. However, after regenerating the resin several times, the level of purity seems to have declined. Has anyone else noticed this with His Select? In general, could someone suggest the typical "lifespan" of His Select or Ni2+ resin in general? What about Glutathione resin? I was also wondering if anyone had experience using cobalt resin? What is the binding capacity of cobalt compared to nickel, and is the selectivity any better than either His-Select or regular Ni-NTA? Also, is it possible to just strip nickel off of the resin and then recharge it with cobalt? Thanks, Matt -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
