Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods
dc.contributor.author | Saumell Esnaola, Miquel | |
dc.contributor.author | Elejaga Jimeno, Ainhoa | |
dc.contributor.author | Echeazarra Escudero, Leire | |
dc.contributor.author | Borrega Román, Leire | |
dc.contributor.author | Barrondo Lacarra, Sergio | |
dc.contributor.author | López de Jesús, Maider | |
dc.contributor.author | González Burguera, Imanol | |
dc.contributor.author | Gomez-Caballero, Alberto | |
dc.contributor.author | Goicolea Altuna, María Aranzazu | |
dc.contributor.author | Sallés Alvira, Joan | |
dc.contributor.author | García del Caño, Gontzal | |
dc.date.accessioned | 2022-11-02T17:33:43Z | |
dc.date.available | 2022-11-02T17:33:43Z | |
dc.date.issued | 2022 | |
dc.identifier.citation | Microbial Cell Factories 21 : (2022) // Article ID 192 | es_ES |
dc.identifier.issn | 1475-2859 | |
dc.identifier.uri | http://hdl.handle.net/10810/58236 | |
dc.description.abstract | Background: Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. Results: Here we generated highly soluble and stable recombinant protein constructs GST-CB1(41)(4-)(472 )and GST-CB1(414-442) containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. Conclusions: Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints. | es_ES |
dc.description.sponsorship | This work was funded by Spanish Ministry of Science, Innovation and Universities (Grant ID, CTQ2017-85686-R) and Basque Government (Research Groups of the Basque University System, Grant IDs, IT1492-22 and IT1620-22). | es_ES |
dc.language.iso | eng | es_ES |
dc.publisher | BMC | es_ES |
dc.rights | info:eu-repo/semantics/openAccess | es_ES |
dc.rights.uri | http://creativecommons.org/licenses/by/3.0/es/ | * |
dc.subject | GPCR expression analysis | es_ES |
dc.subject | quantitative western blot | es_ES |
dc.subject | radioligand saturation binding | es_ES |
dc.subject | cannabinoid CB1 receptor antibodies | es_ES |
dc.subject | carboxy-terminal tail | es_ES |
dc.subject | soluble recombinant protein standards | es_ES |
dc.subject | GST fusion proteins | es_ES |
dc.title | Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods | es_ES |
dc.type | info:eu-repo/semantics/article | es_ES |
dc.rights.holder | © The Author(s) 2022. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. | es_ES |
dc.rights.holder | Atribución 3.0 España | * |
dc.relation.publisherversion | https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-022-01914-1 | es_ES |
dc.identifier.doi | 10.1186/s12934-022-01914-1 | |
dc.departamentoes | Farmacología | es_ES |
dc.departamentoes | Fisiología | es_ES |
dc.departamentoes | Neurociencias | es_ES |
dc.departamentoes | Química analítica | es_ES |
dc.departamentoeu | Farmakologia | es_ES |
dc.departamentoeu | Fisiologia | es_ES |
dc.departamentoeu | Kimika analitikoa | es_ES |
dc.departamentoeu | Neurozientziak | es_ES |
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