Ni-affinity purification and reducing agents

Stephan Witte stephan.witte at
Thu Aug 10 05:58:49 EST 1995

In article <409vtv$88b at>, Per Mygind <perm> wrote:
> -- Fellow Readers
> 	My question to the readers is concerning affinity-purification of recombinant
> proteins, by use of Ni-NTA columns.
> 	I've constructed and purified recombinant proteins of different kinds, but
> I've run my head against a tedious problem. So far, I've used
> beta-mercaptoethanol as a reducing agent, if the fusionprotein in question was
> containing cysteine-residues. One drawback by using this reagent, is that it
> binds covalently to the residues and thereby manipulating an antigenic property
> of the fusionprotein.
> 	My question is, can you use a reducing agent like DTT or DTE instead of
> beta-mercaptoethanol. I've heard that it is not possible with Ni-NTA columns,
> have anyone got experience with that ? 
> Is it maybe possible to remove beta-mercaptoethanol upon purification ? 

strong reducing agents, like DTT, will reduce the Ni-ions, even at
concentrations of 1-5mM, that are required for reducing S-S bonds. My
experiences with mercaptoethanol are quite good. I«ve used concentrations
up to 20 mM successfully.
It can be removed by dialysis, or ultrafiltration or simple size exclusion
chromatography (i.e. Pharmacia PD-10, I like them most)


Stephan Witte
Inst. of Immunology
University of Constance
Postfach 5560 M 660
78434 Konstanz

Phone: +49-7531-88-2749
Fax:     +49-7531-88-3102
e-mail: stephan.witte at

CONSULTANTS: Don«t bore me with telephone calls!  I«ll find you if I want!

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