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
