title: Membrane-SPINE: a biochemical tool to identify protein-protein interactions of membrane proteins in vivo.
abstract: Membrane proteins are essential for cell viability and are therefore important therapeutic targets(1-3). Since they function in complexes(4), methods to identify and characterize their interactions are necessary(5). To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE(6). This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-
  first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.
authors.affiliation: Molekulare Mikrobiologie, Universität Osnabrück.
authors.forename: Volker Steffen
authors.initials: VS
authors.lastname: Müller
authors.forename: Karolin
authors.initials: K
authors.lastname: Tschauner
authors.forename: Sabine
authors.initials: S
authors.lastname: Hunke
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005557:Formaldehyde / Q000737:chemistry; D015151:Immunoblotting / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D016622:Silver Staining / Q000379:methods
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D005557:Formaldehyde; D011494:Protein Kinases; C490475:CpxA protein, E coli