Premium
How to Analyze Protein Complexes by 2D Blue Native SDS‐PAGE
Author(s) -
Reisinger Veronika,
Eichacker Lutz Andreas
Publication year - 2007
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200700205
Subject(s) - proteome , organelle , proteomics , protein subunit , biology , biochemistry , compartmentalization (fire protection) , membrane protein , protein purification , protein subcellular localization prediction , chemistry , membrane , enzyme , gene
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.