antibody crossreactivity problem

Pow Joshi via methods%40net.bio.net (by pow.joshi from gmail.com)
Mon Jul 2 16:39:28 EST 2007


On 7/2/07, Michael Sullivan <mlsulliv from wisc.edu> 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. As many of you probably know, Rubisco is very prominent in
> extracts derived from green plant tissues and can constitute up to
> 50% of the protein in such a sample. Since the antigen was protein
> that was produced in bacteria, I think any cross reactivity must just
> be coincidental.

 You have antibody raised against a peptide ? or just a part of the
recombinant protein?  Either way you could try affinity purification
of your antibody with the protein it was raised against.... you could
have a cleaner antibody.

 Also, the pre-immune serum, did not have any problem
> with rubisco crossreactivity. I haven't found any obvious stretches
> of sequence similarity between rubisco and my protein of interest to
> account for the crossreactivity, but I think it can be hard to make
> predictions about antibody cross-reactivity based on sequence alone.
> Unfortunately, the protein I would like to detect with the antiserum
> is very similar in size to Rubisco. I'm worried I won't be able to
> detect "real" signal from my protein of interest because of the big
> fat Rubisco band.


Another way would be to preadsorb the serum/ antibody with a bacterial
"powder"....  Molecular cloning methods by Sambrook et al or methodss
in molecular biology or even the manual "Antibodies" would give you a
protocol to prepare the bacterial "powder". You don't have to express
the Rubisco there just yet, I imagine.


Best,
pow
>
> Has anybody had a similar problem, and if so, are there any
> solutions? Might different blocking agents help (I'm currently using
> 5% NFDM in TBS)? Can one do a more "stringent" wash to eliminate
> unwanted signals. Currently, I'm thinking the best strategy might be
> to express Rubisco in bacteria and use the lysate to preadsorb the
> antiserum.
>
> Useful comments would be much appreciated.
>
> Mike
>
> ---
> Michael L. Sullivan
> Plant Research Molecular Geneticist
> US Dairy Forage Research Center
> ARS-USDA
> 1925 Linden Drive West
> Madison, WI 53706
> (608) 890-0046 (Phone)
> (608) 890-0076 (FAX)
>
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