[Arabidopsis] Rubisco crossreactivity

Michael Sullivan via arab-gen%40net.bio.net (by mlsulliv from wisc.edu)
Mon Jul 2 11:59:37 EST 2007


I've prepared a rabbit polyclonal antiserum against a protein of  
interest. From immunoblots, it appears that the antiserum is  
crossreacting with the large subunit of Rubisco to make a big fat  
band. 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. Has anybody here tried that strategy before?

Thanks for any input.

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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