An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose

Autores
Zalazar, Lucia; Alonso, Carlos Agustín Isidro; de Castro, Rosana Esther; Cesari, Andreina
Año de publicación
2014
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromise the interaction between the protein and its target. In these cases, GST is a valuable tag providing a long arm that makes the bait protein to be accessible to the mobile flow phase of the chromatography. In the present report, we used a GST-fusion version of the 8 kDa protein SPINK3 as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione agarose beads was modified from previously reported protocols by using an alternative bifunctional crosslinker (DSP) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the re-use of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as bait.
Fil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
Fil: Alonso, Carlos Agustín Isidro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
Fil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
Fil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
Materia
Antibody Purification
Protein-Protein Interactions
Glutathione-Agarose Column
Dsp Cross-Linker
Spink3
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/13285
Acceder
id CONICETDig_5b24923cc9004f20faa3ba57e9fe372c
oai_identifier_str oai:ri.conicet.gov.ar:11336/13285
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agaroseZalazar, LuciaAlonso, Carlos Agustín Isidrode Castro, Rosana EstherCesari, AndreinaAntibody PurificationProtein-Protein InteractionsGlutathione-Agarose ColumnDsp Cross-LinkerSpink3https://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromise the interaction between the protein and its target. In these cases, GST is a valuable tag providing a long arm that makes the bait protein to be accessible to the mobile flow phase of the chromatography. In the present report, we used a GST-fusion version of the 8 kDa protein SPINK3 as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione agarose beads was modified from previously reported protocols by using an alternative bifunctional crosslinker (DSP) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the re-use of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as bait.Fil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; ArgentinaFil: Alonso, Carlos Agustín Isidro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; ArgentinaFil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; ArgentinaFil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; ArgentinaSpringer2014-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/13285Zalazar, Lucia; Alonso, Carlos Agustín Isidro; de Castro, Rosana Esther; Cesari, Andreina; An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose; Springer; Analytical And Bioanalytical Chemistry; 406; 3; 1-2014; 911-9141618-26421618-2650enginfo:eu-repo/semantics/altIdentifier/url/http://link.springer.com/article/10.1007%2Fs00216-013-7533-6info:eu-repo/semantics/altIdentifier/doi/10.1007/s00216-013-7533-6info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-22T11:22:06Zoai:ri.conicet.gov.ar:11336/13285instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-10-22 11:22:06.414CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose
title An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose
spellingShingle An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose
Zalazar, Lucia
Antibody Purification
Protein-Protein Interactions
Glutathione-Agarose Column
Dsp Cross-Linker
Spink3
title_short An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose
title_full An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose
title_fullStr An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose
title_full_unstemmed An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose
title_sort An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose
dc.creator.none.fl_str_mv Zalazar, Lucia
Alonso, Carlos Agustín Isidro
de Castro, Rosana Esther
Cesari, Andreina
author Zalazar, Lucia
author_facet Zalazar, Lucia
Alonso, Carlos Agustín Isidro
de Castro, Rosana Esther
Cesari, Andreina
author_role author
author2 Alonso, Carlos Agustín Isidro
de Castro, Rosana Esther
Cesari, Andreina
author2_role author
author
author
dc.subject.none.fl_str_mv Antibody Purification
Protein-Protein Interactions
Glutathione-Agarose Column
Dsp Cross-Linker
Spink3
topic Antibody Purification
Protein-Protein Interactions
Glutathione-Agarose Column
Dsp Cross-Linker
Spink3
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromise the interaction between the protein and its target. In these cases, GST is a valuable tag providing a long arm that makes the bait protein to be accessible to the mobile flow phase of the chromatography. In the present report, we used a GST-fusion version of the 8 kDa protein SPINK3 as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione agarose beads was modified from previously reported protocols by using an alternative bifunctional crosslinker (DSP) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the re-use of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as bait.
Fil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
Fil: Alonso, Carlos Agustín Isidro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
Fil: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
Fil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina
description Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromise the interaction between the protein and its target. In these cases, GST is a valuable tag providing a long arm that makes the bait protein to be accessible to the mobile flow phase of the chromatography. In the present report, we used a GST-fusion version of the 8 kDa protein SPINK3 as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione agarose beads was modified from previously reported protocols by using an alternative bifunctional crosslinker (DSP) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the re-use of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as bait.
publishDate 2014
dc.date.none.fl_str_mv 2014-01
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/13285
Zalazar, Lucia; Alonso, Carlos Agustín Isidro; de Castro, Rosana Esther; Cesari, Andreina; An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose; Springer; Analytical And Bioanalytical Chemistry; 406; 3; 1-2014; 911-914
1618-2642
1618-2650
url http://hdl.handle.net/11336/13285
identifier_str_mv Zalazar, Lucia; Alonso, Carlos Agustín Isidro; de Castro, Rosana Esther; Cesari, Andreina; An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose; Springer; Analytical And Bioanalytical Chemistry; 406; 3; 1-2014; 911-914
1618-2642
1618-2650
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/http://link.springer.com/article/10.1007%2Fs00216-013-7533-6
info:eu-repo/semantics/altIdentifier/doi/10.1007/s00216-013-7533-6
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Springer
publisher.none.fl_str_mv Springer
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1846781733950717952
score 12.982451

Publicaciones similares