Rubisco crossreactivity

Bean Long via methods%40net.bio.net (by ben.long from yourfinger.anu.edu.au)
Wed Jul 11 22:57:23 EST 2007


Michael,

I know it's been a while since you first asked the question but I might 
be able to offer some help. Rubisco is to the plant proteome what 
albumin is to mammalian systems. There are heaps of Albumin depletion 
kits out there to address exactly this problem. I am sure that someone 
has recently come out with a Rubisco depletion kit, but I can't for the 
life of me find it on-line. Essentially it uses a Rubisco specific 
antibody to deplete your sample before you run it on a gel for westerns. 
Perhaps you could set-up your own depletion kit with a Rubisco Ab. 
Rubisco Ab's can be purchased from a number of companies.

Having said all this, I always have a problem with the Rubisco large 
subunit band cross-reacting with my Ab (to a protein of differing MW). I 
work on cyanobacteria and have a deletion mutant which does not contain 
the gene for the protein I am working on.  I use this mutant to make 
acetone powders which I subsequently use to pre-clear polyclonal serum 
raised against the protein of interest. Theoretically this should give 
me extremely specific polyclonals. Even with this treatment I still get 
cross-reaction to RbcL. We have concluded that this is due to the 
enormous quantity of RbcL in our samples (in my case it constitutes more 
than 80% of total protein in my extracts). We think that the large 
quantities of this protein are, even under electrophoretic conditions, 
dragging the occasional molecule of our protein of interest along with 
it. We have concluded this primarily from the observation that the 
apparent cross-reactivity actually results in what looks like a negative 
RbcL band, with the periphery of the RbcL band taking up most of the Ab. 
This suggests that playing with the electrophoretic conditions (and 
possibly sample buffer) may help too.

Best of luck,

-- 
Bean

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