Affinity Purification

Stephen Gately sg at nwu.edu
Wed Sep 13 16:08:22 EST 1995


We are trying to purify an enzyme from serum-free conditioned medium.  We
have tested a variety of exogenous inhibitors to block the enzyme
activity, and have found a potent inhibitor that is also available bound
to cyanogen bromide activiated agarose.

Despite blocking 100% of activity when the inhibitor is added exogenously,
passing the conditioned medium through a column containing the inhibitor
agarose, or batch adsorption does not block the enzyme activity.

We would appreciate any useful guidelines for affinity purification, ie.
salt, pH etc., or specific comments on why we observe the difference
between exogenous inhibitor and bound inhibitor ability to block
activity.  

Thank you.



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