Recycling Cyanogen bromide activated sepharose affinity columns?
markwell at unlinfo.unl.edu
Fri Jan 28 08:26:33 EST 1994
szsclark at hamlet.ucdavis.edu (Sonya Clark) writes:
>Dear Netters - I am purifying antibodies from a polyclonal prep by
>running them over a CNBr-activated Sepharose affinity column of my
>antigen. As CNBr-activated Sepharose is expensive &/or horrible to make,
>I'd like to strip the column of the antigen currently bound, and re-use
>the sepharose to make another affinity column. Does anyone have a protocol
>to do this? Would eluting the column with harsh eluents (eg.
>Guanidine/urea/SDS) work? and would I then be able to re-use the sepharose
>to couple another antigen? Any advice appreciated. Cheers.
>szsclark at ucd.edu
I don't think that CNBr-Sepharose can be reactivated. As I recall,
the coupling of proteins and other molecules is by forming a covalent
bond, usually to primary amines (e.g. Lys epsilon-amino). After the
antigen, protein, molecule, etc. is coupled, the matrix is treated
with an excess of amino groups to block the remaining active groups.
Thus use of CNBr-Sephadex is a one-shot process. The commercially
available product is expensive, but it is relatively easy to
manufacture in the lab. I hope this helps.
John Markwell Phone: 402-472-2924
Dept. Biochemistry FAX: 402-472-7842
University of Nebraska Internet: markwell at unl.edu
Lincoln, NE 68583-0718
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