Problems w/ native PAGE

[Paul A Bucciaglia]

Hello,

I have been having problems with native PAGE of proteins.  When I extract 
total proteins from tobacco flower organs or leaves (I've listed my 
protocol below) and run them on 4 to 20% denaturing SDS gels, I get fine 
resolution of bands with no detectable smearing.  If I run the same 
amount (30 ug) on a 15% native gel, the bands are poorly resolved and 
smeared along the edges of each lane.  I have also noticed a band of 
precipitated proteins on the interface of the stacking and resolving 
gels; this occurs weather I layer the resolving gel with butanol OR water 
so I don't believe residual butanol is the main problem, as was mentioned 
earlier on the net. I also briefly centifuge the preps after adding 
loading buffer, so I don't believe that particulates are to blame.

Any suggestions out there as to how to improve resoluion of native PAGE?  
Any comments would be much appreciated.

Thanks,

paul bucciaglia



Tobacco Protein Extraction and Electrophoresis

extrac buffer:  50mM KH2PO4, pH 6.8, 14 mM B-ME, 10mM Ascorbate, 1 mM EDTA,
                1 mM EGTA


grind small quantity leaf or anther (0.2-0.5 g) in lN2, add 0.6X 
polyvinyl pyrollidone (insol.), add to an eppendorf tube, add buffer and mix.
Extract 10 mins, spin out insoluble, remove super to another tube.
Ammonium sulfate ppt.
Run over Biogel P-4 spin column (eqilibrated in PO4 buffer).
Quantify protein with Bradford assay

Add equal volume 2x loading buffer (Native, as per Current Protocols in MB)
Spin briefly
Load gel (20ul or less per lane)
Run constant current, 10 mA/Biorad minigel