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Two-dimensional Blue Native/SDS Gel Electrophoresis of Multi-Protein Complexes from Whole Cellular Lysates

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Camacho-Carvajal,  Margarita M.
Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Wollscheid,  Bernd
Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Steimle,  Viktor
Spemann Laboratory, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

Schamel,  Woflgang W. A.
Max Planck Society;

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Citation

Camacho-Carvajal, M. M., Wollscheid, B., Aebersold, R., Steimle, V., & Schamel, W. W. A. (2004). Two-dimensional Blue Native/SDS Gel Electrophoresis of Multi-Protein Complexes from Whole Cellular Lysates. Molecular & Cellular Proteomics, 3, 176-182.


Cite as: http://hdl.handle.net/11858/00-001M-0000-002B-9459-E
Abstract
Identification and characterization of multi-protein complexes is an important step toward an integrative view of protein-protein interaction networks that determine protein function and cell behavior. The limiting factor for identifying protein complexes is the method for their separation. Blue native PAGE (BN-PAGE) permits a high-resolution separation of multi-protein complexes under native conditions. To date, BN-PAGE has only been applicable to purified material. Here, we show that dialysis permits the analysis of multi-protein complexes of whole cellular lysates by BN-PAGE. We visualized different multi-protein complexes by immunoblotting including forms of the eukaryotic proteasome. Complex dynamics after γ interferon stimulation of cells was studied, and an antibody shift assay was used to detect protein-protein interactions in BN-PAGE. Furthermore, we identified defined protein complexes of various proteins including the tumor suppressor p53 and c-Myc. Finally, we identified multi-protein complexes via mass spectrometry, showing that the method has a wide potential for functional proteomics.