Two-dimensional Blue native/SDS gel electrophoresis of multi...

来自 : 发布时间：2024-12-22

ActionsCite Favorites Display options Display options Format Two-dimensional Blue native/SDS GEl electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach 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. Salzano AM, et al. J Proteomics. 2013 Dec 6;94:240-61. doi: 10.1016/j.jprot.2013.09.007. Epub 2013 Sep 20. J Proteomics. 2013. PMID: 24061001 Sunderhaus S, et al. Methods Mol Biol. 2007;372:315-24. doi: 10.1007/978-1-59745-365-3_23. Methods Mol Biol. 2007. PMID: 18314736 Weissman AM. Immunol Today. 1997 Apr;18(4):189-98. doi: 10.1016/s0167-5699(97)84666-x. Immunol Today. 1997. PMID: 9136456 Gerovac M, et al. Front Mol Biosci. 2021 Apr 7;8:661448. doi: 10.3389/fmolb.2021.661448. eCollection 2021. Front Mol Biosci. 2021. PMID: 33898526 Free PMC article. Review. VanAernum ZL, Busch F, Jones BJ, Jia M, Chen Z, Boyken SE, Sahasrabuddhe A, Baker D, Wysocki VH. VanAernum ZL, et al. Nat Protoc. 2020 Mar;15(3):1132-1157. doi: 10.1038/s41596-019-0281-0. Epub 2020 Jan 31. Nat Protoc. 2020. PMID: 32005983 Free PMC article. Pickel C, Günter J, Ruiz-Serrano A, Spielmann P, Fabrizio JA, Wolski W, Peet DJ, Wenger RH, Scholz CC. Pickel C, et al. Redox Biol. 2019 Sep;26:101265. doi: 10.1016/j.redox.2019.101265. Epub 2019 Jul 2. Redox Biol. 2019. PMID: 31299612 Free PMC article. Walker FC, et al. Nucleic Acids Res. 2017 Feb 28;45(4):2112-2123. doi: 10.1093/nar/gkw891. Nucleic Acids Res. 2017. PMID: 28204542 Free PMC article.