Immunoprecipitation...

Kenneth A Frauwirth kfrauwir at mail.med.upenn.edu
Fri Oct 25 12:14:47 EST 2002


On 23 Oct 2002 02:26:40 -0700, FreD 
<f.boal at iecb-polytechnique.u-bordeaux.fr> wrote:
>Hi all!
>
>i am a phd student (bordeaux, France;) and i have some problems with
>an IP. My protein is about 30kDa, and the dimer is about 60kDa. So i
>have some problems to see them on SDS-PAGE (thanks light and heavy IgG
>chains!).
>I can't link my antibodies to proteinG-Sepahrose. 

Have you tried linking your antibody directly to beads?  Dynal sells 
tosyl-activated beads that I've used many times for covalently attaching 
antibodies.

It sounds like you are detecting the IP'ed protein by Western - can you 
radioactively label your protein (35S-Met or 125I)?  Then you can do the 
detection by autoradiography or phosphorimagery, and the antibody won't 
show up.  Alternatively, try finding detection antibodies that will not 
cross-react with the IP antibody (i.e. IP with an antibody from 
one species and detect with an Ab from another, using a secondary that 
does not react with the IP Ab species).

Hope that helps,

Ken Frauwirth

-- 
Ken Frauwirth  (MiSTie #33025)   kfrauwir at mail.med.upenn.edu
Abramson Cancer Research Institute
University of Pennsylvania
http://mail.med.upenn.edu/~kfrauwir
"Science in those days worked in broad strokes.  They got right to the 
point.  Nowadays it's just molecule, molecule, molecule."
                                                     The Tick



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