title: Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis.
abstract: The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.
authors.affiliation: Institute for Medical Virology, University of Zurich; Friedrich Miescher Institute for Biomedical Research.
authors.forename: Patricia E
authors.initials: PE
authors.lastname: Nigg
authors.affiliation: Institute for Medical Virology, University of Zurich; pavlovic.jovan@virology.uzh.ch.
authors.forename: Jovan
authors.initials: J
authors.lastname: Pavlovic
mesh_terms: D000998:Antiviral Agents; D015153:Blotting, Western; D004586:Electrophoresis; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases; D054884:Host-Pathogen Interactions; D006801:Humans; D007372:Interferons; D064547:Myxovirus Resistance Proteins; D054730:Protein Interaction Domains and Motifs
chemical_list: D000998:Antiviral Agents; C579071:MX1 protein, human; D064547:Myxovirus Resistance Proteins; D007372:Interferons; D020558:GTP Phosphohydrolases