Ab purification problem

D.K. dk at no.email.thankstospam.net
Wed Oct 16 22:08:00 EST 2002

ljd3 at duke.edu (Lou D'Amico) wrote:
>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?  

3 mg/ml bound protein is typically optimal for affinity 
chromatography. What was the volume of column used in 
affinity purifucation? 


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