histag elution alternative
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Sat Nov 4 13:22:40 EST 2000
In article <rpg14-2F6424.08251602112000 at nntp-serv.cam.ac.uk>, "Richard P.
Grant" <rpg14 at yahoo.co.uk.invalid> wrote:
: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.
I thought it was crystal clear. His message
><l2bct8.h9j.ln at wpxx02.toxi.uni-wuerzburg.de>:
>DTT will turn your column into a dirty brownish colour.
>Given that it is quite unclear what happens there (reduction,
>but to what? I would guess that elementary Nickel is unlikely
>to form), I would go with the EDTA elution.
>A resin treated with DTT is almost impossible to recharge
>(I know since I tried :-).
So, there was Ni2+-NTA, which was reduced with DTT to brownish
stuff (Ni+, I'd guess), then he tried to recharge it. Recharging
_must_ include stripping Ni away!
: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?
Well, it's still chemical. One fantasy would be that reduced Ni+ might
catalyze some hydrolytic reaction, though... _If_ DTT treated Ni-NTA
cannot be recharged, I'd seriously consider something along these lines.
But, enough theories. Here is an experiment. I took an old column that
needs to be regenerated anyway, filled it with 200 mM DTT in TBS
and in 30 min it turned dark brown-orange. Good, here we have the
reduction. Now the only thing that remains is to remove this Ni+ and
recharge with Ni2+. Surprisingly, it turned out to be _very_ difficult:
- EDTA does not elute brown stuff at all (only traces of Ni2+ were eluted
judged by characteristic blue color of Ni2+-EDTA chelates).
- 6 M GuCl does not elute it (my standard regeneration step).
- 1 M NaOH does not elute.
- 1 M acetate does not elute it (no incubation, just gravity flow
- 50 mM NiSO4 does not displace brown stuff either.
What holds colorless stuff in the column, I have no idea. Either NTA
has very high affinity for Ni+ or whatever form the Ni+/Ni is there, it
is insoluble and "sticks" to agarose. In other words, Cornelius was
absolutely correct - "A resin treated with DTT is almost impossible to
recharge". The significant correction, however, would be "...almost
impossible to _strip_". Once it is done, recharging is no problem:
1 M HCl, upon long "soaking" incubation for 1 Hr at room temp (totally
OK for crosslinked agarose), decolorizes the column turning it into
normal colorless/white agarose. The eluate is apparently colorless too.
At this point addition of 50 mM NiSO4 after H20 wash to remove acid
turns the column into normal blue color. In other words, either HCl
eluted Ni or turned it into form that is easily displaced by Ni2+. Like
I suspected, there are no any irreversible changes brought upon by DTT.
Chemistry is my weakest point so I am not sure about the exact reaction
involved at every step.
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