title: Preparation of Gap Junctions in Membrane Microdomains for Immunoprecipitation and Mass Spectrometry Interactome Analysis.
abstract: Protein interaction networks at gap junction plaques are increasingly implicated in a variety of intracellular signaling cascades. Identifying protein interactions of integral membrane proteins is a valuable tool for determining channel function. However, several technical challenges exist. Subcellular fractionation of the bait protein matrix is usually required to identify less abundant proteins in complex homogenates. Sufficient solvation of the lipid environment without perturbation of the protein interactome must also be achieved. The present chapter describes the flotation of light and heavy liver tissue membrane microdomains to facilitate the identification and analysis of endogenous gap junction proteins and includes technical notes for translation to other integral membrane proteins, tissues, or cell culture models. These procedures are valuable tools for the enrichment of gap junction membrane compartments and for the identification of gap junction signaling interactomes.
authors.affiliation: Neural Regeneration Laboratory, Ottawa Institute of Systems Biology, Centre for Catalysis Research and Innovation, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON, Canada. slfwlr@gmail.com.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Fowler
authors.affiliation: Neural Regeneration Laboratory, Ottawa Institute of Systems Biology, Centre for Catalysis Research and Innovation, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON, Canada.
authors.forename: Mark
authors.initials: M
authors.lastname: Akins
authors.affiliation: Neural Regeneration Laboratory, Ottawa Institute of Systems Biology, Centre for Catalysis Research and Innovation, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON, Canada.
authors.forename: Steffany A L
authors.initials: SA
authors.lastname: Bennett
mesh_terms: D053000:Analytic Sample Preparation Methods / Q000379:methods*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002458:Cell Fractionation; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D047468:Immunoprecipitation / Q000379:methods*; D008099:Liver / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry / Q000379:methods*; D000097002:Gap Junction beta-1 Protein
chemical_list: D017630:Connexins; D000097002:Gap Junction beta-1 Protein
keywords: Cx32; Endogenous immunoprecipitation; Gap junctions; Integral membrane protein; Interactome; Membrane fractionation; Membrane microdomains; Network analysis; Opti-Prep gradient; Proteomics