How to analyze protein complexes by 2D blue native SDS-PAGE.
Natural compartmentalization makes proteome analysis of the cell, cell organelles and organelle subfractions possible. Protein complexes are the basis for the next level of compartmentalization that can be addressed well with proteomic technology. Protein complexes organize and maintain the cellular and organelle functions on all levels of complexity in time and space. Cell development and division, transcription and translation, respiration and photosynthesis, transport and metabolism can be defined by the activity of protein complexes. Since a large part of the protein complexes of the cell body are inserted in lipid membrane phases, isolation, separation and protein subunit identification were difficult to address. Blue native polyacrylamide gel electrophoresis (BN-PAGE) provides us with the technology for high resolution separation of membrane protein complexes. Here, we show that high resolution separation of protein complexes by BN-PAGE requires the establishment of a detailed solubilisation strategy. We show that BN/SDS-PAGE provides the scientist with a high resolution array of protein subunits which allows analysis of the specific subunit stoichiometry of a protein complex as well as the assembly of protein complexes by standard protein detection methodology like DIGE, gelblot analysis and mass spectrometry. We envision BN-PAGE to precede classical 2D IEF/SDS-analysis for detailed characterization of membrane proteomes.
Reisinger
Veronika
V
Ludwig Maximilian University, Munich, Germany.
Eichacker
Lutz Andreas
LA
D002451:Cell Compartmentation; D004072:Digitonin / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005960:Glucosides / Q000494:pharmacology; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000302:isolation & purification*; D008823:Micelles; D046912:Multiprotein Complexes / Q000032:analysis*; D012394:Rosaniline Dyes; D012995:Solubility; D020524:Thylakoids / Q000737:chemistry
D005960:Glucosides; D008565:Membrane Proteins; D008823:Micelles; D046912:Multiprotein Complexes; D012394:Rosaniline Dyes; C040358:dodecyl maltoside; C048139:Coomassie blue; D004072:Digitonin