Kenneth A Frauwirth kfrauwir at
Fri Oct 25 12:14:47 EST 2002

On 23 Oct 2002 02:26:40 -0700, FreD 
<f.boal at> 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
>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 

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
Abramson Cancer Research Institute
University of Pennsylvania
"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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