histag elution alternative
ffrank at rz.uni-potsdam.de
Sun Nov 5 09:06:04 EST 2000
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote:
>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.
I would expect that it is Ni°-metal, which is of course poorly soluble
in anything, and which sticks to the column material because upon
reduction, the metal starts crystallizing there, not in the free
>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.
HCl oxidizes the Ni° (to Ni2+, I think) (while H+ is reduced to H2).
The question remains why you don't see the Nickel - perhaps it was du
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