Ab purification problem

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Wed Oct 16 21:48:16 EST 2002


My experience with ABs isn't as significant as I'd like it to be, however
here's a few comment/questions:

Could it be that you have generated an antibody to an epitope that is
normally hidden - i.e. part of your antibody is targeting peptides that
aren't normally exposed so your (mostly folded) affinity reagent isn't
working ? How long is the linker between your target protein and the matrix
? Can you determine what is your actual epitope ? Is it continuous or
discontinuous ?

A.G.E.

"Lou D'Amico" <ljd3 at duke.edu> wrote in message
news:3dadb7d6.3968236 at news.duke.edu...
> 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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