Help! Protein oxidation on Nickel column!

Eric C. Anderson anderson at pharmdec.wustl.edu
Thu Jun 15 05:38:18 EST 1995


In article <3rkdbm$irs at osceola.cs.ucf.edu>, barry at fhis.net (Barry
Schweitzer) wrote:
> 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. 

unless you can't add BME to your column run for a reason related directly
to your protein, you should add it to your column binding/washing and all
elution buffers.  while DTT will destroy the column, BME has no ill
effects on the column.  i use it at 5mM and it seems to remove any
disulfide bonding problems.

good luck,

eric

-- 
"i don't know what caffeine does for you, but i'm pretty sure that without it your head caves in."

eric c. anderson                                 anderson at pharmdec.wustl.edu
dept. of molecular bio. and pharm.               (314)362-3963 (lab)
washington univ. school of medicine              (314)362-7058 (FAX)
660 s. euclid box 8103                           
st. louis, mo 63110

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