Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Autor(en): Mueller, Volker Steffen
Tschauner, Karolin
Hunke, Sabine 
Stichwörter: Bioengineering; formaldehyde cross-linking; IDENTIFICATION; in vivo protein-protein interaction; Issue 81; MASS-SPECTROMETRY; Membrane Proteins; MS-analysis; Multidisciplinary Sciences; Science & Technology - Other Topics; Strep-tag
Erscheinungsdatum: 2013
Herausgeber: JOURNAL OF VISUALIZED EXPERIMENTS
Journal: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Ausgabe: 81
Zusammenfassung: 
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-SPINE6. 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-SPINE: 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.
ISSN: 1940087X
DOI: 10.3791/50810

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