Ab purification problem

D.K. dk at no.email.thankstospam.net
Thu Oct 17 21:28:19 EST 2002


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

Hardly matters. With 60K antigen, polyclonal antibody population
and random chemical coupling to the column, there would be 
all kind of combinations. 

DK

>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
>>
>>
>



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