Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis.
We report a subdivision of the mitochondrial proteome into defined sets of proteins, which is based on the combination of three different gel electrophoresis procedures. First, Blue-native polyacrylamide gel electrophoresis is employed to separate mitochondrial protein complexes. The protein complexes are electroeluted and completely detached from Coomasssie blue. Subsequently the subunits of the protein complexes are separated by isoelectric focusing and finally by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The resolution capacity of the procedure is demonstrated for the ATP synthase complex, the cytochrome c reductase complex and the preprotein translocase of the outer mitochondrial membrane (the TOM complex). The method allows the separation of isoforms of subunits forming part of protein complexes, whose occurrence seems to be rather a rule than an exception in higher eukaryotes. Furthermore, extremely hydrophobic proteins are detectable on the gels.
Werhahn
Wolf
W
Institut für Angewandte Genetik, Universität Hannover, Hannover, Germany.
Braun
Hans-Peter
HP
D000251:Adenosine Triphosphatases / Q000032:analysis; D029681:Arabidopsis Proteins / Q000032:analysis*; D001426:Bacterial Proteins; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D029968:Escherichia coli Proteins / Q000032:analysis; D057927:Hydrophobic and Hydrophilic Interactions; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins / Q000032:analysis; D024101:Mitochondrial Proteins / Q000032:analysis*; D025261:Mitochondrial Proton-Translocating ATPases / Q000032:analysis; D009245:NADH Dehydrogenase / Q000032:analysis; D010940:Plant Proteins / Q000032:analysis; D021122:Protein Subunits; D020543:Proteome / Q000032:analysis; D012394:Rosaniline Dyes; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D010940:Plant Proteins; D021122:Protein Subunits; D020543:Proteome; D012394:Rosaniline Dyes; D000069816:SEC Translocation Channels; C048139:Coomassie blue; D009245:NADH Dehydrogenase; D000251:Adenosine Triphosphatases; D025261:Mitochondrial Proton-Translocating ATPases; D000081416:SecA Proteins