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
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/13285
Ver los metadatos del registro completo
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network_name_str |
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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 Técnicas2025-09-29T09:52:43Zoai: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:34982025-09-29 09:52:43.449CONICET 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 |
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1844613615444819968 |
score |
13.070432 |