@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}},
}

Altmetric

View in Altmetric

Web of Science

Times cited: