antibody crossreactivity problem

Michael Sullivan via methods%40net.bio.net (by mlsulliv from wisc.edu)
Mon Jul 2 12:03:27 EST 2007


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. 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.

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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