Removing high pI proteins

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Fri Apr 30 10:20:57 EST 2004


Ben Long wrote:

> Hi all,
> 
> I have a protein extract containing a complex I am interested in but have
> found (by LC/MS analysis) that it is highly contaminated with ribosomal
> proteins with very high pI's (around 12).  I believe they are well and truly
> stuck to my complex and wanted to remove them somehow without disrupting my
> complex too much.  Any ideas??

Separation itself could be achieved by ion exchange chromatography,
given the high charge of the contaminants at physiological pH. But if
the contaminants bind to your proteins, you have to dissociate the
unwanted, but retain the wanted protein-protein interactions. This might
be possible by carefully raising the denaturant concentration (nonionic
denaturant like urea).

Mild ionic denaturants like diiodosalycilate may also be used, but would
require a separation method different from ion exchange chromatography. 

The other alternative would be complete dissociation of the complex,
purification of the individual components and renaturation. 

Which of these routes is possible in your case is impossible to predict,
certainly from the info given. 



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