Help! Protein oxidation on Nickel column!

Barry Schweitzer barry at fhis.net
Tue Jun 13 11:11:34 EST 1995


Hello Netters:

We are overexpressing a 21 kD His-tagged protein, and purifying the
protein using a Nickel column.  
Light scattering and molecular seive chromatography suggest that a 
significant amount of the "pure" material is a dimer.   
Further dimerization  occurs if we don't add B-ME immediately following
the column purification.  Reductants cannot be used while running the 
column. The protein contains 3 cysteine residues but no disulfides.
Our working hypothesis is that one of the cysteins exposed on the 
surface is forming a disulfide bridge between another molecule due
to oxidation while the crude extract is on the nickel column.  This
column runs fairly slowly. 

Anybody have any suggestions on how we might be able to prevent this
oxidation?

Thanks in advance! 
 
--
Barry Schweitzer, Ph.D.
Director                                     Assistant Professor
Division of Structural Biology               Department of Chemistry
Walt Disney Memorial Cancer Institute        University of Central Florida
 at Florida Hospital
12722 Research Parkway
Orlando, FL 32826
Phone:	(407) 380-9977
FAX:	(407) 380-9978
email:	barry at sneezy.fhis.net



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