Connoisseurs of Ni-NTA

Dr. Artem Evdokimov eudokima at mail.ncifcrf.gov
Mon May 14 10:02:30 EST 2001


Hi,

Have you (can you ?) checked oligomeric homogeneity before the Ni-NTA
step ? If you have some non-paired cysteines or you have some reduced
protein then you can have normal multimers not related to nickel.

Nickel-sulfur chelation is indeed possible and is very strong. One way
to avoid it would be to use very small amounts of thiol reagents, not
enough to reduce your disulphides but enough to kill off all exposed
nickel. Alternatively, you could pre-wash your column with e.g. 5 mM BME
and then extensively wash with water. I had one case where the protein
had exposed S-S bonds (total of 5 S-S pairs in the protein) and was a
bloody mess because of assorted crosslinking. I have never definitevely
proven that nickel was 100% to blame because several multimeric forms
were also observed before the Ni-NTA step.

If you have access to MS, you can detect nickel 'bridges' quite easily -
they do not normally decompose under ESI conditions.

Hope it helps,

Artem

Michael Witty wrote:
> 
> Here is one for connoisseurs of Ni-NTA.  When I loaded a protein (22kDa)
> onto Ni-NTA, I eluted, roughly, 22, 44, 66kDa.  This protein has
> disulphide bonds and I suspect some kind of bad breaking of disulphide
> bonds and reforming between molecules.  Does anyone have similar
> experience of this kind of thing?  Mike.

-- 
|Dr. Artem Evdokimov   Protein Engineering |
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