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Immunology and Infection

Co-Immunopräzipitation der Maus Mx1 Protein mit dem Influenza A Virus Nucleoprotein

Published: April 21st, 2015

DOI:

10.3791/52871

1Inflammation Research Center, VIB, 2Department of Biomedical Molecular Biology, Ghent University

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and 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.

Myxovirus Widerstand (Mx) Proteine ​​sind ein wichtiger Teil der angeborenen Immunabwehr gegen virale Pathogene. Diese Proteine ​​sind groß dynamin artigen GTPasen, die durch Typ I und Typ III Interferone induziert werden. Die entsprechenden Mx Gene in fast allen Wirbeltieren vorhanden sind in einer oder mehreren Kopien und deren Genprodukte hemmen ein breites Spektrum von Viren, einschließlich Orthomyxoviridae (z. B. Influenza-Virus), Rhabdoviridae (z. B. vesikuläres Stomatitis-Virus), Bunyaviridae (z. , La Crosse Virus) und Retroviridae (zB Human Immunodeficiency Virus-1) 1-4. Es ist unklar, wie diese Proteine ​​erkenn....

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Hinweis: Die nach der Transfektion und Co-Immunopräzipitation Protokoll wird für einen 9 cm Petrischale Format etabliert. Andere Formate sind auch nach der Skalierung des Protokolls möglich.

1. Seeding die humane embryonale Nierenzellen (HEK) 293T-Zellen

  1. Samen der HEK293T Zellen einen Tag vor der Transfektion mit 1,2 x 10 6 Zellen pro 9 cm Petrischale in 12 ml Dulbeccos modifiziertem Eagle-Medium (DMEM) mit 10% fötalem Kälberserum, 2 mM L-Glutamin, 0,4 mM Natriumpy.......

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N-Ethylmaleimid ist eine organische Verbindung, die verwendet werden können, um irreversibel ändern freien Thiolgruppen, beispielsweise zur Cysteinproteasen (Figur 1) zu hemmen.

Die antivirale Mx1-Protein hemmt die Influenza-A-Virus-Replikation durch die Interaktion mit dem viralen Nukleoprotein. Die hier beschriebene optimierte Co-Immunopräzipitation-Protokoll ermöglicht, diese NP-Mx1 Interaktion zu studieren. HEK293T-Zellen wurden mit Expressionsvektoren für .......

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Untersuchung der Wechselwirkung zwischen der antiviralen Proteine ​​und ihre virale Ziele ist sehr wichtig, um die Details des antiviralen Mechanismus dieser Proteine ​​zu verstehen. Dies kann neue Erkenntnisse darüber, wie Viren und ihren Wirten co-entwickelt geben und die Grundlage für die Entwicklung neuer antiviraler Strategien. Die hier beschriebene optimierte Coimmunpräzipitation Protokoll ermöglicht, die Interaktion zwischen der Maus Mx1-Protein und seine virales Ziel, das Influenza-NP-Protein zu unte.......

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Diese Arbeit wurde von FWO-Vlaanderen, der IOF Projekt IOF10 / StartT / 027 und der Universität Gent Sonderforschungsstipendium BOF12 / GOA / 014 unterstützt.

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NameCompanyCatalog NumberComments
DMEM high glucoseGibco52100-047
N-EthylmaleimideSigmaE-3876Toxic
Igepal CA-630SigmaI-30212also known as NP40
Protease Inhibitor CocktailRoche11 873 580 001
anti-NP monoclonal antibodyNIH Biodefense and Emerging Infections Research Resources RepositoryNR-4282ascites blend of clones A1 and A3
anti-RNP polyclonal serumNIH Biodefense and Emerging Infections Research Resources RepositoryNR-3133directed against A/Scotland/840/74 (H3N2)
Protein G Sepharose 4FFGE Healthcare17-0618-01
Hyperfilm ECL 18 x 24 cmGE Healthcare28-9068-36
ECL Western Blotting SubstratePierce32106

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