Media problems on Nickel column
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Fri Jan 31 10:56:31 EST 1997
In article <cmf5-3001971657440001 at 126.96.36.199>, cmf5 at cornell.edu (Casey
M. Finnerty) wrote:
#My problem is that sample loading causes leaching of the nickel off the
#column. My equilibration buffer alone does not cause noticeable nickel
#leaching, so I don't think the imidazole is a problem.
#I've tried dialyzing the sample into the equilibration buffer, and this
#helped but did not eliminate the problem (I may have not fully dialyzed
#the sample, however).
#Some have suggested that I switch to a NTA based resin, but I have
#trouble believing that a tetradentate chelator will be all that much
#better than a tridentate chelator such as IDA. Am I wrong?
Yes, you are. Not only NTA is "better" (binds Ni stronger), it is also
more specific toward His as opposed to other aromatics.
#I am thinking
#that I may need to concentrate and buffer exchange my sample, though I'm
#worried that too much concentration may cause precipitation of the
#surfactants or serum proteins in the medium (and perhaps co-precipitation
#of my recombinant protein). Dialysis bags will not be practical, as I hope
#to scale up to > 1 L.
It's impractical to even load 1 L! Here is a perfect solution: do ammonium
sulphate precipitation (will take a separate day to determine proper %
cut), resuspend pellet in loading buffer, load. Loading sample will be in
a higher salt than loading buffer but that's fine - IMAC "likes" high
salt. This will i) get rid of whatever causes the leaching (if dialysis
indeed helped as you say), ii) reduce volume, iii) increase purity, iv)
allow to use less resin.
And yes, switch to NTA.
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