antibody crossreactivity problem

Tom Anderson via methods%40net.bio.net (by ucgatan from ucl.ac.uk)
Mon Jul 2 13:24:33 EST 2007


On Mon, 2 Jul 2007, Michael Sullivan wrote:

> I've prepared a rabbit polyclonal antiserum against a plant protein of
> interest. From immunoblots, it appears that the antiserum is
> crossreacting with the large subunit of Rubisco to make a big fat band.

Ouch!

> Might different blocking agents help (I'm currently using
> 5% NFDM in TBS)? Can one do a more "stringent" wash to eliminate
> unwanted signals.

You might as well try BSA as your block, and using a carbonate instead of
tris-glycine buffer for the transfer, but these are usually tricks to
reduce nonspecific binding, rather than a cross-reaction. I've never
mucked about with stringency of washes, but more detergent apparently does
the trick; i would imagine higher salt might too. Again, though, if you
have a specific cross-reaction, i'd guess this probably won't help.

> Currently, I'm thinking the best strategy might be to express Rubisco in
> bacteria and use the lysate to preadsorb the antiserum.

Sadly, it might be. Or if you happened to have plant cells that didn't
express your protein, you could pre-adsorb against lysate from those,
which would avoid having to make rubisco in bugs.

I guess making a monoclonal, and picking one that doesn't cross-react, is
also an option, but a rather laborious one.

Alternatively, if you're currently running denaturing PAGE, maybe try a
native gel; if you're lucky, the epitope that's being recognised on
rubisco will be hidden, and the one on your protein won't. It should also
shift rubisco up away from your band, since it's a monstrous huge complex.
Of course, if your protein is also in a complex, this may not be so great!

tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson from ucl.ac.uk



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