title: Optimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome.
abstract: 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.
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21224, USA.
authors.forename: Lesley A
authors.initials: LA
authors.lastname: Kane
authors.forename: Christina K
authors.initials: CK
authors.lastname: Yung
authors.forename: Giulio
authors.initials: G
authors.lastname: Agnetti
authors.forename: Irina
authors.initials: I
authors.lastname: Neverova
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Van Eyk
mesh_terms: 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*
chemical_list: D020543:Proteome