Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach.
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 gamma 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.
Camacho-Carvajal
Margarita M
MM
Max Planck-Institute of Immunobiology, Stübeweg 51, 79108 Freiburg, Germany.
Wollscheid
Bernd
B
Aebersold
Ruedi
R
Steimle
Viktor
V
Schamel
Wolfgang W A
WW
D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D019169:Jurkat Cells; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D040901:Proteomics; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
C489427:MYC protein, human; D009097:Multienzyme Complexes; C489428:Myc protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D016159:Tumor Suppressor Protein p53; D007371:Interferon-gamma; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex