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High MW IgG signals in IP/co-IP with protein G

IgG Rabbit IgG Co-IP IP non-specific bands

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#1 miST32

miST32

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Posted 19 February 2014 - 01:04 PM

Hi all,

I've been struggling with a co-IP for months and need some assistance.  Namely, I'm detecting high molecular weight bands in my IgG IP control.  I add 5ug of normal rabbit IgG (same as IP antibody), and perform the immunoprecipitation with protein G dynabeads.  When I western blot, I get distinct bands at ~50, 75, and ~120 kDa with both light-chain specific HRP, and True-blot (a secondary reagent that only recognizes native IgG, supposedly).  These bands make it very hard to tell if I get IP of target proteins ~60kDa and 140 in my actual sample lanes.

Standard SDS-PAGE and transfer to nitrocellulose.  Nothing special.  

Has anybody else had a tough time with normal rabbit IgG in regards to these high molecular weight species on western blot?  A colleague in my lab also had the same trouble with this experiment.  
 

I've detected similar bands (though not as robust) with BS3 cross-linking of the antibody or IgG to the dynabeads, so I'm wondering if this might be non-reduced/denatured or cross-linked protein-G/IgG complex eluting from the beads.  Very annoying if so!

Any thoughts?  I wold be happy to provide more info as needed.

Cheers and thanks!
 



#2 miST32

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Posted 01 March 2014 - 08:50 PM

Just in case anybody else runs into this problem - I figured it out.  Embarrassingly simple problem, actually.  rolleyes.gif  

Turns out I was using insufficient reducing agent (an old/expired bottle).  The high molecular weight signal was almost certainly nothing more than non-reduced IgG, as it cleared up with a new stock and higher concentration in my samples.

Cheers







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