An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP

Descripción del Articulo

Here, we present an in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of split-GFP for translation test (FAST), based on the small fragment GFP11 binding to GFP1-10fast. We detail the expression and purification of the GFP1-10fast protein, DNA template amplificati...

Descripción completa

Detalles Bibliográficos
Autores: Quiroz-Huanca, Ana, Sanchez-Castro, Ana, Soriano-Castillo, Pablo, Poletti, Chiara, Nloh Tientcheu, Therese Manuela, Fabbretti, Attilio, Giuliodori, Anna Maria, Milon, Pohl
Formato: artículo
Fecha de Publicación:2024
Institución:Universidad Peruana de Ciencias Aplicadas
Repositorio:UPC-Institucional
Lenguaje:inglés
OAI Identifier:oai:repositorioacademico.upc.edu.pe:10757/683804
Enlace del recurso:http://hdl.handle.net/10757/683804
Nivel de acceso:acceso embargado
Materia:Biotechnology and bioengineering
Chemistry
High-Throughput Screening
Protein expression and purification
id UUPC_11b09e59c68192b1a068b5c3f6ff3b5f
oai_identifier_str oai:repositorioacademico.upc.edu.pe:10757/683804
network_acronym_str UUPC
network_name_str UPC-Institucional
repository_id_str 2670
dc.title.es_PE.fl_str_mv An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP
title An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP
spellingShingle An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP
Quiroz-Huanca, Ana
Biotechnology and bioengineering
Chemistry
High-Throughput Screening
Protein expression and purification
title_short An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP
title_full An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP
title_fullStr An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP
title_full_unstemmed An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP
title_sort An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP
author Quiroz-Huanca, Ana
author_facet Quiroz-Huanca, Ana
Sanchez-Castro, Ana
Soriano-Castillo, Pablo
Poletti, Chiara
Nloh Tientcheu, Therese Manuela
Fabbretti, Attilio
Giuliodori, Anna Maria
Milon, Pohl
author_role author
author2 Sanchez-Castro, Ana
Soriano-Castillo, Pablo
Poletti, Chiara
Nloh Tientcheu, Therese Manuela
Fabbretti, Attilio
Giuliodori, Anna Maria
Milon, Pohl
author2_role author
author
author
author
author
author
author
dc.contributor.author.fl_str_mv Quiroz-Huanca, Ana
Sanchez-Castro, Ana
Soriano-Castillo, Pablo
Poletti, Chiara
Nloh Tientcheu, Therese Manuela
Fabbretti, Attilio
Giuliodori, Anna Maria
Milon, Pohl
dc.subject.es_PE.fl_str_mv Biotechnology and bioengineering
Chemistry
High-Throughput Screening
Protein expression and purification
topic Biotechnology and bioengineering
Chemistry
High-Throughput Screening
Protein expression and purification
description Here, we present an in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of split-GFP for translation test (FAST), based on the small fragment GFP11 binding to GFP1-10fast. We detail the expression and purification of the GFP1-10fast protein, DNA template amplification, in vitro GFP11-tagged CspA synthesis, FAST detection of the GFP11-tagged protein, and optional recovery of the fluorescent complex. In vitro synthesis of GFP11 maximizes the molar yield of synthesized proteins, providing enhanced sensitivity to test translation inhibitors. For complete details on the use and execution of this protocol, please refer to Pham et al.
publishDate 2024
dc.date.accessioned.none.fl_str_mv 2025-01-07T14:17:10Z
dc.date.available.none.fl_str_mv 2025-01-07T14:17:10Z
dc.date.issued.fl_str_mv 2024-12-20
dc.type.es_PE.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.doi.none.fl_str_mv 10.1016/j.xpro.2024.103448
dc.identifier.uri.none.fl_str_mv http://hdl.handle.net/10757/683804
dc.identifier.eissn.none.fl_str_mv 26661667
dc.identifier.journal.es_PE.fl_str_mv STAR Protocols
dc.identifier.eid.none.fl_str_mv 2-s2.0-85209135433
dc.identifier.scopusid.none.fl_str_mv SCOPUS_ID:85209135433
dc.identifier.pii.none.fl_str_mv S2666166724006130
dc.identifier.isni.none.fl_str_mv 0000 0001 2196 144X
dc.identifier.ror.none.fl_str_mv 047xrr705
identifier_str_mv 10.1016/j.xpro.2024.103448
26661667
STAR Protocols
2-s2.0-85209135433
SCOPUS_ID:85209135433
S2666166724006130
0000 0001 2196 144X
047xrr705
url http://hdl.handle.net/10757/683804
dc.language.iso.es_PE.fl_str_mv eng
language eng
dc.rights.es_PE.fl_str_mv info:eu-repo/semantics/embargoedAccess
eu_rights_str_mv embargoedAccess
dc.format.es_PE.fl_str_mv application/html
dc.publisher.es_PE.fl_str_mv Cell Pres
dc.source.es_PE.fl_str_mv Universidad Peruana de Ciencias Aplicadas (UPC)
Repositorio Academico - UPC
dc.source.none.fl_str_mv reponame:UPC-Institucional
instname:Universidad Peruana de Ciencias Aplicadas
instacron:UPC
instname_str Universidad Peruana de Ciencias Aplicadas
instacron_str UPC
institution UPC
reponame_str UPC-Institucional
collection UPC-Institucional
dc.source.journaltitle.none.fl_str_mv STAR Protocols
dc.source.volume.none.fl_str_mv 5
dc.source.issue.none.fl_str_mv 4
bitstream.url.fl_str_mv https://repositorioacademico.upc.edu.pe/bitstream/10757/683804/1/license.txt
bitstream.checksum.fl_str_mv 8a4605be74aa9ea9d79846c1fba20a33
bitstream.checksumAlgorithm.fl_str_mv MD5
repository.name.fl_str_mv Repositorio académico upc
repository.mail.fl_str_mv upc@openrepository.com
_version_ 1846066137215795200
spelling ff83a51976577e1fa98b6b36ae02678e300a353e43d44abe60a513c007b2d563dfdc8c169600c306233d00b857c6326bfe141903a074b5cc0e3eda42f011cda70d7500a15ae12ac83a3300a544a4ba1122ca11ce7d3677fe3a72ddb74f3ce87f443fde500fa113616c9458917c7f80380c760c3da5003df0fb6ba04b8d92158878fbe8da7e1a500Quiroz-Huanca, AnaSanchez-Castro, AnaSoriano-Castillo, PabloPoletti, ChiaraNloh Tientcheu, Therese ManuelaFabbretti, AttilioGiuliodori, Anna MariaMilon, Pohl2025-01-07T14:17:10Z2025-01-07T14:17:10Z2024-12-2010.1016/j.xpro.2024.103448http://hdl.handle.net/10757/68380426661667STAR Protocols2-s2.0-85209135433SCOPUS_ID:85209135433S26661667240061300000 0001 2196 144X047xrr705Here, we present an in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of split-GFP for translation test (FAST), based on the small fragment GFP11 binding to GFP1-10fast. We detail the expression and purification of the GFP1-10fast protein, DNA template amplification, in vitro GFP11-tagged CspA synthesis, FAST detection of the GFP11-tagged protein, and optional recovery of the fluorescent complex. In vitro synthesis of GFP11 maximizes the molar yield of synthesized proteins, providing enhanced sensitivity to test translation inhibitors. For complete details on the use and execution of this protocol, please refer to Pham et al.Horizon 2020Revisión por paresapplication/htmlengCell Presinfo:eu-repo/semantics/embargoedAccessUniversidad Peruana de Ciencias Aplicadas (UPC)Repositorio Academico - UPCSTAR Protocols54reponame:UPC-Institucionalinstname:Universidad Peruana de Ciencias Aplicadasinstacron:UPCBiotechnology and bioengineeringChemistryHigh-Throughput ScreeningProtein expression and purificationAn in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFPinfo:eu-repo/semantics/articleLICENSElicense.txtlicense.txttext/plain; charset=utf-81748https://repositorioacademico.upc.edu.pe/bitstream/10757/683804/1/license.txt8a4605be74aa9ea9d79846c1fba20a33MD51false10757/683804oai:repositorioacademico.upc.edu.pe:10757/6838042025-01-07 14:17:12.262Repositorio académico upcupc@openrepository.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
score 13.945474
An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP
Nota importante:
La información contenida en este registro es de entera responsabilidad de la institución que gestiona el repositorio institucional donde esta contenido este documento o set de datos. El CONCYTEC no se hace responsable por los contenidos (publicaciones y/o datos) accesibles a través del Repositorio Nacional Digital de Ciencia, Tecnología e Innovación de Acceso Abierto (ALICIA).

Ejemplares Similares