Ab purification problem

[Lou D'Amico]

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
my problem!

-Lou D'Amico


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
>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
>
>