electrophoresis of plant proteins
paul-b at molbio.cbs.umn.edu
Thu Dec 9 18:46:06 EST 1993
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 tube. 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
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