Protocol for Immuno-Enrichment of FLAG-Tagged Protein Complexes

This protocol describes immunoprecipitation of proteins associated with FLAG-tagged recombinant proteins followed by mass spectrometry-based proteomics to identify the associated interactome components. FLAG epitope was chosen, because existing high-affinity monoclonal antibodies allow for sensitive...

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Autores principales: Valdez-Sinon, Arielle Nicole, Gokhale, Avanti, Faundez, Victor, Bassell, Gary Jonathan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2020
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580096/
https://www.ncbi.nlm.nih.gov/pubmed/33111116
http://dx.doi.org/10.1016/j.xpro.2020.100083
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author Valdez-Sinon, Arielle Nicole
Gokhale, Avanti
Faundez, Victor
Bassell, Gary Jonathan
author_facet Valdez-Sinon, Arielle Nicole
Gokhale, Avanti
Faundez, Victor
Bassell, Gary Jonathan
author_sort Valdez-Sinon, Arielle Nicole
collection PubMed
description This protocol describes immunoprecipitation of proteins associated with FLAG-tagged recombinant proteins followed by mass spectrometry-based proteomics to identify the associated interactome components. FLAG epitope was chosen, because existing high-affinity monoclonal antibodies allow for sensitive immunoprecipitation and FLAG peptides permit efficient elution of protein complexes. With many commercially available FLAG tools, this protocol is highly versatile. This procedure reduces immunoprecipitation of nonspecific binding proteins. Gene ontology analyses performed following mass spectrometry-based proteomics may elucidate novel functions of proteins of interest. For complete details on the use and application of this protocol, please refer to Valdez-Sinon et al. (2020).
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spelling pubmed-75800962020-10-26 Protocol for Immuno-Enrichment of FLAG-Tagged Protein Complexes Valdez-Sinon, Arielle Nicole Gokhale, Avanti Faundez, Victor Bassell, Gary Jonathan STAR Protoc Protocol This protocol describes immunoprecipitation of proteins associated with FLAG-tagged recombinant proteins followed by mass spectrometry-based proteomics to identify the associated interactome components. FLAG epitope was chosen, because existing high-affinity monoclonal antibodies allow for sensitive immunoprecipitation and FLAG peptides permit efficient elution of protein complexes. With many commercially available FLAG tools, this protocol is highly versatile. This procedure reduces immunoprecipitation of nonspecific binding proteins. Gene ontology analyses performed following mass spectrometry-based proteomics may elucidate novel functions of proteins of interest. For complete details on the use and application of this protocol, please refer to Valdez-Sinon et al. (2020). Elsevier 2020-08-11 /pmc/articles/PMC7580096/ /pubmed/33111116 http://dx.doi.org/10.1016/j.xpro.2020.100083 Text en © 2020 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Valdez-Sinon, Arielle Nicole
Gokhale, Avanti
Faundez, Victor
Bassell, Gary Jonathan
Protocol for Immuno-Enrichment of FLAG-Tagged Protein Complexes
title Protocol for Immuno-Enrichment of FLAG-Tagged Protein Complexes
title_full Protocol for Immuno-Enrichment of FLAG-Tagged Protein Complexes
title_fullStr Protocol for Immuno-Enrichment of FLAG-Tagged Protein Complexes
title_full_unstemmed Protocol for Immuno-Enrichment of FLAG-Tagged Protein Complexes
title_short Protocol for Immuno-Enrichment of FLAG-Tagged Protein Complexes
title_sort protocol for immuno-enrichment of flag-tagged protein complexes
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580096/
https://www.ncbi.nlm.nih.gov/pubmed/33111116
http://dx.doi.org/10.1016/j.xpro.2020.100083
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