To separate protein from Ni+2/EDTA
AEVDOKIMOZ at cinci.rr.com
Wed Jan 15 01:37:38 EST 2003
> Sounds strange. One would think that EDTA is in large excess and
> since it binds Ni with Ka > 10e13 there would be no Ni bound to
Yes, except for Ni bound to S and especially in places where a convenient S
is next to other residues forming a cage, or several protein molecules
aggregate around some Ni ions and form an impenetrable greasy barrier of
'stuff'. These combinations can beat even EDTA. In one case, we had (MS
evidence) Ni ions trailing after the protein was incubated with 50 mM EDTA.
In yet one more case Ni ions persisted even after denaturation !
The evidence is fuzzy, but in more than one case when the Ni-IMAC bound
protein was eluted with EDTA it was not good at all - yet when we purified
it using other techniques (excluding the IMAC even) - it would be OK. It is
not impossible to imagine that some proteins just don't like Ni-NTA, or
sometimes if a protein can't be eluted from Ni-NTA it means that the
irreversibly bound portion is compromised :)
All I meant was - the original poster should not be upset if the
Ni-incubated protein is not happy anymore.
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