(by ben.long from yourfinger.anu.edu.au)
Wed Jul 11 22:57:23 EST 2007
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,
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