Recycling Cyanogen bromide activated sepharose affinity columns?

john markwell 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.

>Sonya Clark.
>Botany Dept.,
>UCDavis
>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

--
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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