Ab purification problem

Lou D'Amico ljd3 at duke.edu
Wed Oct 16 14:07:50 EST 2002


I'm trying to affinity purify an AB i generated in guinea pig to an
insect enzyme (JH-esterase).  It's approximately 60kD.  When we had a
company originally do the affinity purification, the yield was low
(maybe 500 ug out of 20mls of serum).  The serum that passed through
the column was still chock full of reactive Ab (confirmed by Western
Blot).  I did another purification in house on an IgG purified extract
of the serum, and still got low yields in the purification (maybe
300ug).  I don't have tons of the antigen to work with, and I was
curious if someone could help me with two questions.  
1) Does the size of the antigen impact yield this severly?  I was
coupling 2-3mg on a ml of column matrix.
2)  Are there standard alternative techniques to purifying Ab's of
large mol weight proteins that might be more appropriate?  

Thanks in advance for any information you can provide, either here, or
to ljd3 at duke.edu.

All best,
Lou D'Amico

Department of Biology
Duke University



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