Optimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome.
Separation of basic proteins with 2-DE presents technical challenges involving protein precipitation, load limitations, and streaking. Cardiac mitochondria are enriched in basic proteins and difficult to resolve by 2-DE. We investigated two methods, cup and paper bridge, for sample loading of this subproteome into the basic range (pH 6-11) gels. Paper bridge loading consistently produced improved resolution of both analytical and preparative protein loads. A unique benefit of this technique is that proteins retained in the paper bridge after loading basic gels can be reloaded onto lower pH gradients (pH 4-7), allowing valued samples to be analyzed on multiple pH ranges.
Kane
Lesley A
LA
Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21224, USA.
Yung
Christina K
CK
Agnetti
Giulio
G
Neverova
Irina
I
Van Eyk
Jennifer E
JE
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008929:Mitochondria, Heart / Q000737:chemistry*; D010449:Peptide Mapping; D020543:Proteome / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013367:Submitochondrial Particles / Q000737:chemistry*
D020543:Proteome