Help! Protein oxidation on Nickel column!
barry at fhis.net
Tue Jun 13 11:11:34 EST 1995
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
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
More information about the Methods