Ab purification problem
ljd3 at duke.edu
Thu Oct 17 13:07:52 EST 2002
Thanks for the thoughts. I'm not sure i can answer all of the
questions, so you gave me something to think about!
The antigen injected into the guinea pig was native, and had activity
in vitro. I suppose it could be orienting on the matrix in a way
that's hiding the recognized epitope, but i thought with a large
antigen, and a polyclonal antiserum i'd get a cocktail of recognizing
Ab's that hit on multiple epitopes with varying specificities... a
couple of the companies i talked to said i didnt need a spacer between
the matrix and the target protein because of the size of the target
protein. That said, I don't have any idea what actually is the
epitope, and if it's continuous or discontinuous.
Does that make anything else come to mind? Thanks for thinking about
On Thu, 17 Oct 2002 02:48:16 GMT, "Artem Evdokimov"
<AEVDOKIMOZ at cinci.rr.com> wrote:
>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
>"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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