[Arabidopsis] Follow up: Rubisco on immunoblots
(by mlsulliv from wisc.edu)
Thu Aug 9 11:04:30 EST 2007
A few weeks ago, I posted a query regarding cross reactivity of
antiserum with Rubisco on western blots. From the responses I got, it
is clear this is a pretty widespread phenomenon. For me, this was
especially problematic, because my protein of interest is very
similar in size to Rubisco. I thought a follow up of some of my
results might be useful to those who have had similar problems.
Several people suggested affinity purification of the antibody would
help. I did this and it did help cut down on the Rubisco cross-
reacting signal significantly, but not completely. Again, because my
protein of interest has a predicted migration so close to Rubisco, I
still had concerns my real signal might be obscured.
Jim Tokuhisa from Virginia Tech suggested using polyethyleneimine
(PEI) to deplete extracts of Rubisco. PEI is often used in
purification of nucleic acid binding proteins because it is
relatively selective in what it precipitates, but it also seems to
precipitate Rubisco. The original protocol Jim sent me did not work
as written for our extracts. I suspect this had to do with some
differences in the buffer composition we use in making extracts.
However, I had forwarded the protocol to Barb Moffatt who said, at
least from stained gels, the protocol worked well in her lab. My
postdoc did do some additional fooling around with the protocol, as
well as talking about it with Dick Burgess, whose lab did some of the
original work using PEI for protein purification.
What Dick suggested to us is that we should empirically determine the
PEI precipitation parameters. Consequently, we have made plant
extracts our usual way, then tried precipitation with various
concentrations of PEI (from a 10% pH 8.0 stock solution). We have
indeed found that depending on how extracts are made, different
amounts of PEI might be needed. E.g., my postdoc made extracts one
way and got good precipitation of Rubisco with 0.1% PEI. I use a
slightly different extraction buffer to prevent PPO-mediated
oxidative browning and found that my extracts required more PEI
(0.2%) for precipitation: 0.1% really failed to precipitate any
Rubisco for me, but 0.2% worked really well, with near quantitative
precipitation of Rubisco large subunit. I did not really see much
apparent change in the protein composition of the remaining extract
as judged by Coomassie staining of an SDS-PAGE gel. Interestingly, in
my experiment, for some extracts, higher levels of PEI (0.4%) left
more Rubisco in solution.
If anybody is interested in trying this, I can send you Jim's
original protocol if you e-mail me. Alternatively, you might simply
try making an extract with a pH 7.5-8 Tris-based buffer. I usually
use 3 ml buffer per g fresh weight. Once the extract is made, make
aliquots with various final concentrations of PEI-- 0, 0.1, 0.2, 0.4%
is what I used in my experiment. The solution will become immediately
turbid. Put on ice for 5 min, then spin at about 7000 g for 5 min.
Remove the supernatant. You can then prepare the supernatant samples
for SDS-PAGE. Note that samples with more than 0.2% or so PEI may
form a precipitate with sample buffer. I just spun this out prior to
gel loading. If you are interested in what is precipitated by PEI, my
understanding (I haven't tried this) is that proteins, but not
nucleic acids, will be resolubilized by resuspending the PEI pellet
in buffer with 1 M NaCl.
In my experiment, 0.2% PEI nearly quantitatively precipitated Rubisco
from my extract. This allowed me to visualize a band on a subsequent
western. Prior to the precipitation, this band was not apparent due
to the Rubisco interference.
The caveat for all this is there is no guarantee PEI won't
precipitate the protein you are interested in. However, it's a
simple, relatively inexpensive procedure, so it might be worth giving
a try if you are having "Rubisco issues".
I hope you all find this helpful.
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)
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