Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein.
Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP-Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.
Inflammation Research Center, VIB; Department of Biomedical Molecular Biology, Ghent University.
Judith
J
Verhelst
Inflammation Research Center, VIB; Department of Biomedical Molecular Biology, Ghent University.
Dorien
D
De Vlieger
Inflammation Research Center, VIB; Department of Biomedical Molecular Biology, Ghent University; xavier.saelens@vib-ugent.be.
Xavier
X
Saelens
D000818:Animals; D005033:Ethylmaleimide / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D009980:Influenza A virus / Q000737:chemistry; D051379:Mice; D064547:Myxovirus Resistance Proteins / Q000737:chemistry*; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins / Q000737:chemistry*; D014758:Viral Core Proteins / Q000737:chemistry*
C579070:Mx1 protein, mouse; D064547:Myxovirus Resistance Proteins; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D005033:Ethylmaleimide