electrophoresis of plant proteins

[Mark Kalous]

In article <CHsL2C.H7F at news2.cis.umn.edu> paul-b at molbio.cbs.umn.edu (Paul Bucciaglia) writes:
>From: paul-b at molbio.cbs.umn.edu (Paul Bucciaglia)
>Subject: electrophoresis of plant proteins
>Date: Thu, 9 Dec 1993 23:48:39 GMT

>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.

Here's something that might help.  We've used tobacco leaves and its worked 
quite well.  to the frozen powder add: 0.0625 M Tris + 2% SDS +10% glycerol 
ph6.8  and as a reductant use dithiothriotol 50 microM (doesn't streak as much 
as mercapto-ethanol when run in PAGE)  Boil samples 5 min to solubilize all 
protein. Centrifuge. Use protein sample as quickly as possible and minimize 
freeze-thaw.

Mark
e-mail: mkalous at pnfi.forestry.ca


>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