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Target protein in flow through, wash, and all fractions following heparin IEX chromatography

James A. Dutko jd4300 at albany.edu
Tue Jul 8 10:39:36 EST 2003


I am attempting to purify a protein complex containing several 
subunits.  One subunit has an affinity tag that I use to track the complex 
throughout the extraction by western blot.  In a pilot experiment using 
POROS resins from Perseptive Biosystems, we found that the complex bound to 
heparin and cation resins but not to anion resin.  Some fraction of the 
material was in the flow through during those experiments, the wash was not 
analyzed, and the complex was eluted in the 100-300 mM NaCl range with no 
leakiness below or above.  We selected the heparin column since there 
seemed to be less in the flow through.  The problem I have encountered 
subsequently with the heparin is the complex seems to be in every fraction, 
wash, and flow through.  The westerns are unambiguous, negative and 
positive controls are fine.  The western blots may be more sensitive than 
in the pilot experiments since I switched to loading beads rather than 
loading eluted material.  However, I do not see a significant increase in 
signal in the 100-300 mM range compared to the wash.  We have not altered 
the extraction procedure or buffers from previous runs.  Does the complex 
actually bind heparin or is it just washing over the column?  Thanks for 
any ideas or explanations.

James

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