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

Judith Verhelst (UGent) , Dorien De Vlieger (UGent) and Xavier Saelens (UGent)
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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.
Keywords
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

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Citation

Please use this url to cite or link to this publication:

Chicago
Verhelst, Judith, Dorien De Vlieger, and Xavier Saelens. 2015. “Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein.” Jove-journal of Visualized Experiments (98).
APA
Verhelst, J., De Vlieger, D., & Saelens, X. (2015). Co-immunoprecipitation of the mouse Mx1 protein with the influenza A virus nucleoprotein. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS, (98).
Vancouver
1.
Verhelst J, De Vlieger D, Saelens X. Co-immunoprecipitation of the mouse Mx1 protein with the influenza A virus nucleoprotein. JOVE-JOURNAL OF VISUALIZED EXPERIMENTS. 2015;(98).
MLA
Verhelst, Judith, Dorien De Vlieger, and Xavier Saelens. “Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein.” JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 98 (2015): n. pag. Print.
@article{6973338,
  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.},
  articleno    = {e52871},
  author       = {Verhelst, Judith and De Vlieger, Dorien and Saelens, Xavier},
  issn         = {1940-087X},
  journal      = {JOVE-JOURNAL OF VISUALIZED EXPERIMENTS},
  keywords     = {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},
  language     = {eng},
  number       = {98},
  pages        = {7},
  title        = {Co-immunoprecipitation of the mouse Mx1 protein with the influenza A virus nucleoprotein},
  url          = {http://dx.doi.org/10.3791/52871},
  year         = {2015},
}

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