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Co-immunoprecipitation of the mouse Mx1 protein with the influenza A virus nucleoprotein

By Judith Verhelst, Dorien De Vlieger and Xavier Saelens

Abstract

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

Topics: Medicine and Health Sciences, nucleoprotein, Influenza A virus, Issue 98, Immunology, Mx1, co-immunoprecipitation, N-ethylmaleimide, plasmid transfection, viral infection, antiviral host defense, HIV-1 INFECTION, N-ETHYLMALEIMIDE, THOGOTO, GTPASE, NUCLEOCAPSIDS, SENSITIVITY, INHIBITOR, COMPLEX
Publisher: 'MyJove Corporation'
Year: 2015
DOI identifier: 10.3791/52871
OAI identifier: oai:archive.ugent.be:6973338
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