Problems w/ native PAGE
Paul A Bucciaglia
bucc0003 at gold.tc.umn.edu
Mon Oct 24 10:21:19 EST 1994
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.
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)
Load gel (20ul or less per lane)
Run constant current, 10 mA/Biorad minigel
More information about the Methods