histag elution alternative
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Wed Nov 1 18:21:35 EST 2000
In article <rpg14-35EC13.20351901112000 at news.ic24.net>, "Richard P. Grant" <rpg14 at yahoo.co.uk.invalid> wrote:
:In article <8tpqc8$kna$1 at news.doit.wisc.edu>,
:klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote:
:> Interesting... While I do not dispute the finding (never tried
:> myself), I am puzzled. Why would it be so??? There simply
:> isn't anything in NTA-agarose that can be reduced by DTT.
:> NTA is coupled to agarose through spacer via AFAIR epoxy
:> chemistry (not affected by DTT), NTA itself is basicaly three
:> acetates linked together. Sepharose is just a crosslinked
:> agarose, so it is not affected by DTT either.
:Hang on. Where's the nickel?
The nickel is gone. We are talking about _recharging_ and that
necessarily implies stripping the original, fully or partially reduced,
stuff. So the only things left are agarose, NTA, and linker arm. I am
interested to learn what else is there that might respond to DTT
or to learn how would these things be different after DTT treatment.
:I'm guessing that Ni3+ or Ni2+ gets
:converted to Nickel metal (or Ni1+) in the process we're talking about.
:(Reduction is electron gain, recalling some chemistry from a long time
:ago (-: ). Which would bugger up your acetate chelation, big time.
:And although the only reference I have at home does not give the colours
:of the oxidation states, I wouldn't be surprised if blue -> murky brown
:is consistent with Ni3+ -> Ni1+/0
:> Anyone wants to bet? I say that reduced Ni-NTA agarose
:> can be stripped and recharged w/o a problem.
:So, 'While I do not dispute the finding' you do, actually think that
:Cornelius *and* the manufacturer are either wrong or lying?
Neither. I do not dispute Cornelius' finding. Evidently, that's what
he got. I have not seen any similar manufacturer's claims. To the
contrary, they say that after several runs with a lot of E.coli
stuff, the column may turn brownish (can't be any different since
reduction potential of E.coli cytosol is very high), in which case it
_can_ be stripped and recharged.
I do however _doubt_ Cornelius' finding (see the difference?).
Granted, I do it a priori, based only on theoretical considerations for
now (generally, not a very smart thing to do :-)). But I _do_ want to
know if there _really_ is a problem described and if there is, I am
certainly very interested to learn _why_. Betting on it does not
necessarily help but I also don't see how it hurts. I am an adventurous
type and I bet regularly on outcome of this and that experiement with
however is willing to.
There, Richard, I suggest a book under $20 from amazone.com? :-))
More information about the Proteins