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
> 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.
"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
for a comprehensive list of bike related web pages... http://pharmdec.wustl.edu/~anderson/anderson.html
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