protein-protein interaction (very strong)

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


In article <73lh6b$cle$1 at mserv2.dl.ac.uk>, nieto <nieto at porthos.bio.ub.es>
wrote:

> 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 porthos.bio.ub.es

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
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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