protein-protein interaction (very strong)

Bernard P. Murray, PhD bpmurray*STUFFER* at
Fri Nov 27 15:41:53 EST 1998

In article <73lh6b$cle$1 at>, nieto <nieto at>

> Dear friends,
> By means of a His-tag I heve detectected a strong protein-protein 
> interaction.
> The complex is eluted from the Ni-colum at 100 mM imidazol.The 
> interaction is resistant up to 2M KCl. Both proteins separate in SDS-PAGE 
> without B-mercaproethanol or boiling. 
> Any suggestions of what can be going on here? Any nondenaturing ways of 
> disrupting the interaction?
> Thank you all.
> jo
> nieto at

Just to play the devil's advocate...  Is there any chance that
your non-His-tagged protein in the "complex" can stick to the
Ni-column independently?  I just want to be sure that you are
looking at a protein-protein interaction instead of simple
co-purification.  Do you have any evidence, independent of the
Ni-column, that the proteins interact?  Certainly if your
putative interaction partner contains a di-His I would be
worried about this.
     This being said, if you look at texts/reviews on
immunopurification you should find lists of ways of disrupting
strong interactions.  I don't have anything to hand at the
moment but I seem to remember that high concentrations of
MgCl2 or potassium thiocyanate were recommended or maybe
you could include organic solvents (eg. acetone).
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA

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