histag elution alternative

Dima Klenchin 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

Absolutely! 

:> 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? :-))

        - Dima






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