Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly...

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Detalles Bibliográficos
Autores: Morató Arús, Xavier, López-Cano, Marc, Canas, Paula M., Cunha, Rodrigo A, Ciruela Alférez, Francisco
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2017
País:España
Institución:Universidad de Barcelona
Repositorio:Dipòsit Digital de la UB
OAI Identifier:oai:diposit.ub.edu:2445/213380
Acceso en línea:https://hdl.handle.net/2445/213380
Access Level:acceso abierto
Palabra clave:Cervell
Detergents
Proteïnes de membrana
Ratolins (Animals de laboratori)
Brain
Membrane proteins
Mice (Laboratory animals)
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oai_identifier_str oai:diposit.ub.edu:2445/213380
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network_name_str España
repository_id_str
spelling Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein LocalizationMorató Arús, XavierLópez-Cano, MarcCanas, Paula M.Cunha, Rodrigo ACiruela Alférez, FranciscoCervellDetergentsProteïnes de membranaRatolins (Animals de laboratori)BrainDetergentsMembrane proteinsMice (Laboratory animals)Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.JoVE2017info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://hdl.handle.net/2445/213380Articles publicats en revistes (Patologia i Terapèutica Experimental)reponame:Dipòsit Digital de la UBinstname:Universidad de BarcelonaInglésReproducció del document publicat a: https://doi.org/10.3791/55661JoVE. Journal of Visualized Experiments, 2017, num.123https://doi.org/10.3791/55661(c) JoVE, 2017info:eu-repo/semantics/openAccessoai:diposit.ub.edu:2445/2133802026-05-27T06:46:51Z
dc.title.none.fl_str_mv Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization
title Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization
spellingShingle Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization
Morató Arús, Xavier
Cervell
Detergents
Proteïnes de membrana
Ratolins (Animals de laboratori)
Brain
Detergents
Membrane proteins
Mice (Laboratory animals)
title_short Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization
title_full Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization
title_fullStr Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization
title_full_unstemmed Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization
title_sort Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization
dc.creator.none.fl_str_mv Morató Arús, Xavier
López-Cano, Marc
Canas, Paula M.
Cunha, Rodrigo A
Ciruela Alférez, Francisco
author Morató Arús, Xavier
author_facet Morató Arús, Xavier
López-Cano, Marc
Canas, Paula M.
Cunha, Rodrigo A
Ciruela Alférez, Francisco
author_role author
author2 López-Cano, Marc
Canas, Paula M.
Cunha, Rodrigo A
Ciruela Alférez, Francisco
author2_role author
author
author
author
dc.subject.none.fl_str_mv Cervell
Detergents
Proteïnes de membrana
Ratolins (Animals de laboratori)
Brain
Detergents
Membrane proteins
Mice (Laboratory animals)
topic Cervell
Detergents
Proteïnes de membrana
Ratolins (Animals de laboratori)
Brain
Detergents
Membrane proteins
Mice (Laboratory animals)
description Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.
publishDate 2017
dc.date.none.fl_str_mv 2017
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv https://hdl.handle.net/2445/213380
url https://hdl.handle.net/2445/213380
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Reproducció del document publicat a: https://doi.org/10.3791/55661
JoVE. Journal of Visualized Experiments, 2017, num.123
https://doi.org/10.3791/55661
dc.rights.none.fl_str_mv (c) JoVE, 2017
info:eu-repo/semantics/openAccess
rights_invalid_str_mv (c) JoVE, 2017
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv JoVE
publisher.none.fl_str_mv JoVE
dc.source.none.fl_str_mv Articles publicats en revistes (Patologia i Terapèutica Experimental)
reponame:Dipòsit Digital de la UB
instname:Universidad de Barcelona
instname_str Universidad de Barcelona
reponame_str Dipòsit Digital de la UB
collection Dipòsit Digital de la UB
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,811543

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