histag elution alternative
Richard P. Grant
rpg14 at yahoo.co.uk.invalid
Thu Nov 2 03:25:16 EST 2000
In article <8tq8il$8nq$1 at news.doit.wisc.edu>,
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote:
> The nickel is gone. We are talking about _recharging_ and that
> necessarily implies stripping the original, fully or partially reduced,
>From Cornelius' posting, that's not that crystal clear. I'm not sure
whether he reduced it in the nickel-bound state or not. It would be
interesting to take some resin, treat it with EDTA (which seems to strip
the nickel), then add DTT. And, of course, compare with non-EDTA
treated. It looks like in the presence of DTT the nickel is reduced
(well, duh) to a state that not just makes the Ni2+ fall off (because in
the absence of DTT it can be recharged with NiCl2) but causes something
else to happen - maybe Ni+ is strange, hmmm what about Ni-?? (thinking
aloud here). The other thing is, given that the nickel is resistant to
10 mM bMe, and your point about the reducing potential of the cell,
maybe the change wrought by DTT is chemical rather than oxidative?
Sulphonation, perhaps? Maybe someone who has time to waste (!) could
check some redox potentials . . .
> stuff. So the only things left are agarose, NTA, and linker arm. I am
NTA is 'what' triacetate? I mean, what exactly is there?
Just looked it up. Nitrilotriacetic acid. Hmm. What's that nitrogen
> :> Anyone wants to bet? I say that reduced Ni-NTA agarose
> :> can be stripped and recharged w/o a problem.
Just noticed this again - you seemed to be talking about the resin in
the presence of nickel, unless the above was just a typo?
> he got. I have not seen any similar manufacturer's claims. To the
We got our wires crossed there; I apologize.
> 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.
<aside - Qiagen touting the reusability of their system? wow, it must
As above, this points to a chemical reaction . . . maybe?
> There, Richard, I suggest a book under $20 from amazone.com? :-))
Mwah hah hah. As if post-docs over here aren't skint enough :-/
I've a better idea. I'll suggest some experiments and you can report
back with the findings. :) Alternatively, we kidnap a Qiagen scientist
and, eh, *extract* the information we need. :-))
Richard P. Grant MRC Lab of Mol Biol
rpg 'at' mrc-lmb.cam.ac.uk http://www2.mrc-lmb.cam.ac.uk/personal/rpg/
'I think I speak for everyone here when I say, "Huh?"'
More information about the Proteins