electrophoresis of plant proteins

[Paul Bucciaglia]

Sorry if this has already been posted here but it was returned to me
this evening.....

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