[Arabidopsis] Follow up: Rubisco on immunoblots

Michael Sullivan via arab-gen%40net.bio.net (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.

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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