electrophoresis of plant proteins

[RossN at imb.lan.nrc.ca]

I have just started working on algal proteins and have had some similar
SDS-PAGE experiences which I have attributed (rightly?) to the contamination
of my samples with acidic polysaccharides.  For one gel I centrifuged my
sample (13,000g x 5 min) prior to loading and got better resolution.  I also
saw a visible opaque pellet in the microtube after spinning which I guessed
was polysaccharide.
    Another possibility is that smearing is due to a high concentration of
lipids.  Lipids can be removed from the sample by extracting your plant
material with ether/ethanol (3/2) 3X (I used this for brain myelin
delipidation, 7-8 yrs ago).  Acetone or chloroform/methanol (2/1) might also
work.
    Good luck.   Neil
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From: paul-b at molbio.cbs.umn.edu (Paul Bucciaglia)
Subject: electrophoresis of plant proteins
Date: 9 Dec 93 23:48:39 GMT
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Hello folks,
 
I have been unhappy with the results of my plant  protein PAGE gels for
some time now and am looking for some suggestions.  Let me start by stating
what is working--electrophoresis of bacterial extracts or even pelleted cells
or bugs straight out of culture boiled in loading buffer and electrophoresed
on 4 --> 20 % gradient gels.  this results in a crisp banding pattern when
the gels are staine with Coomassie.
 
I have been less succesful with protien extracts from leaves or stamens
of tobacco.  Usually I grind the tissue under lN2 in a mortar and pestle
and add the frozen powder to tris (200mM pH 7.8) plus 14mM beta mercapto
ethanol.  The extracts are microcetrifuged and the supers removed to another
tub
e.  I add an equal amonunt of 2x load buffer (current proteocals in
mol. biol recipe), boil 3', cool and load 10-20 microliters/well under
the same buffer conditions that work great for bacterial proteins.
 
The resulting gels lack the resolution and sharpness that I see in proteins
from bacteria run under the same conditons on the same gels.
 
So any suggestions?  do I need to further purify my crude extracts of
plant tissue?  Are there any compounds which might cause the smearing
and lack of resolution that I see in plant extracts? Any thoughts would
be appreciated.
 
paul bucciaglia