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Characterization of multi-subunit protein complexes of human mxa using non-denaturing polyacrylamide gel-electrophoresis


Nigg, P E; Pavlovic, J (2016). Characterization of multi-subunit protein complexes of human mxa using non-denaturing polyacrylamide gel-electrophoresis. Journal of Visualized Experiments (Jove), 116:online.

Abstract

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

Abstract

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Medical Virology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > General Neuroscience
Physical Sciences > General Chemical Engineering
Life Sciences > General Biochemistry, Genetics and Molecular Biology
Life Sciences > General Immunology and Microbiology
Uncontrolled Keywords:Oligomerization, non-denaturing polyacrylamide gel electrophoresis (PAGE), western blot analysis, myxovirus resistance A (MxA) protein, multi-subunit protein complexes, quaternary structure
Language:English
Date:28 October 2016
Deposited On:23 Jan 2017 14:40
Last Modified:31 Jul 2020 00:10
Publisher:Journal of Visualized Experiments
ISSN:1940-087X
Funders:Grant from the Swiss National Science foundation (Grant nr. 31003A_143834) to JP
OA Status:Green
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.3791/54683
PubMed ID:27842357
Project Information:
  • : FunderSNSF
  • : Grant ID
  • : Project TitleGrant from the Swiss National Science foundation (Grant nr. 31003A_143834) to JP

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