Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14)
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Revista Brasileira de Zootecnia (Online) |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-35982020000100407 |
Resumo: | ABSTRACT The objective of the present study was to develop a methodology to obtain the enriched fraction of ram seminal vesicle protein 14 (RSVP14). The study was developed using Morada Nova rams, from which semen samples were collected weekly. Seminal plasma proteins were precipitated with cold ethanol, and then 6.15 mg/mL of total proteins were subjected to liquid gelatin affinity chromatography using a Gelatin-Sepharose matrix coupled to an automated chromatographic system. Proteins were eluted into four fractions (A, B, C, and D), in which A and B contained non-gelatin-binding proteins, and C and D fractions contained gelatin-binding proteins. Gels were analyzed by Quantity One software, in which five protein bands were detected in fraction D, with molecular weights between 12 and 30 kDa. The gelatin-binding proteins (fraction D) were loaded into a HiTrap™ Heparin HP affinity column. Two chromatographic fractions were separated (D1 and D2), in which D1 contained non-heparin-binding proteins, and D2 contained heparin-binding proteins. Proteins from the last two peaks were subjected to 12.5% SDS-PAGE and Western Blot. Two bands with molecular weight of 14 and 24 kDa, contained in fraction D1, were excised from gel and subjected to tandem mass spectrometry, identifying the proteins RSVP14 and RSVP24. Thus, the chromatographic methods of the present study are efficient to capture the enriched fraction of RSVP14. |
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Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14)Ovis ariesproteomicsspermatozoaABSTRACT The objective of the present study was to develop a methodology to obtain the enriched fraction of ram seminal vesicle protein 14 (RSVP14). The study was developed using Morada Nova rams, from which semen samples were collected weekly. Seminal plasma proteins were precipitated with cold ethanol, and then 6.15 mg/mL of total proteins were subjected to liquid gelatin affinity chromatography using a Gelatin-Sepharose matrix coupled to an automated chromatographic system. Proteins were eluted into four fractions (A, B, C, and D), in which A and B contained non-gelatin-binding proteins, and C and D fractions contained gelatin-binding proteins. Gels were analyzed by Quantity One software, in which five protein bands were detected in fraction D, with molecular weights between 12 and 30 kDa. The gelatin-binding proteins (fraction D) were loaded into a HiTrap™ Heparin HP affinity column. Two chromatographic fractions were separated (D1 and D2), in which D1 contained non-heparin-binding proteins, and D2 contained heparin-binding proteins. Proteins from the last two peaks were subjected to 12.5% SDS-PAGE and Western Blot. Two bands with molecular weight of 14 and 24 kDa, contained in fraction D1, were excised from gel and subjected to tandem mass spectrometry, identifying the proteins RSVP14 and RSVP24. Thus, the chromatographic methods of the present study are efficient to capture the enriched fraction of RSVP14.Sociedade Brasileira de Zootecnia2020-01-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-35982020000100407Revista Brasileira de Zootecnia v.49 2020reponame:Revista Brasileira de Zootecnia (Online)instname:Sociedade Brasileira de Zootecnia (SBZ)instacron:SBZ10.37496/rbz4920190166info:eu-repo/semantics/openAccessFelix,Bruna AlvesOtávio,Kamila de SousaMartins,Jorge André MatiasSantos,Fágner Cavalcante Patrocínio dosVelho,Ana Luiza Malhado Cazaux de SouzaVasconcelos,Fábio RogerBezerra,Maria Júlia BarbosaMoura,Arlindo A.eng2020-10-28T00:00:00Zoai:scielo:S1516-35982020000100407Revistahttps://www.rbz.org.br/pt-br/https://old.scielo.br/oai/scielo-oai.php||bz@sbz.org.br|| secretariarbz@sbz.org.br1806-92901516-3598opendoar:2020-10-28T00:00Revista Brasileira de Zootecnia (Online) - Sociedade Brasileira de Zootecnia (SBZ)false |
dc.title.none.fl_str_mv |
Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14) |
title |
Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14) |
spellingShingle |
Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14) Felix,Bruna Alves Ovis aries proteomics spermatozoa |
title_short |
Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14) |
title_full |
Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14) |
title_fullStr |
Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14) |
title_full_unstemmed |
Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14) |
title_sort |
Methods for obtaining the enriched fraction of ram seminal vesicle proteins (RSVP14) |
author |
Felix,Bruna Alves |
author_facet |
Felix,Bruna Alves Otávio,Kamila de Sousa Martins,Jorge André Matias Santos,Fágner Cavalcante Patrocínio dos Velho,Ana Luiza Malhado Cazaux de Souza Vasconcelos,Fábio Roger Bezerra,Maria Júlia Barbosa Moura,Arlindo A. |
author_role |
author |
author2 |
Otávio,Kamila de Sousa Martins,Jorge André Matias Santos,Fágner Cavalcante Patrocínio dos Velho,Ana Luiza Malhado Cazaux de Souza Vasconcelos,Fábio Roger Bezerra,Maria Júlia Barbosa Moura,Arlindo A. |
author2_role |
author author author author author author author |
dc.contributor.author.fl_str_mv |
Felix,Bruna Alves Otávio,Kamila de Sousa Martins,Jorge André Matias Santos,Fágner Cavalcante Patrocínio dos Velho,Ana Luiza Malhado Cazaux de Souza Vasconcelos,Fábio Roger Bezerra,Maria Júlia Barbosa Moura,Arlindo A. |
dc.subject.por.fl_str_mv |
Ovis aries proteomics spermatozoa |
topic |
Ovis aries proteomics spermatozoa |
description |
ABSTRACT The objective of the present study was to develop a methodology to obtain the enriched fraction of ram seminal vesicle protein 14 (RSVP14). The study was developed using Morada Nova rams, from which semen samples were collected weekly. Seminal plasma proteins were precipitated with cold ethanol, and then 6.15 mg/mL of total proteins were subjected to liquid gelatin affinity chromatography using a Gelatin-Sepharose matrix coupled to an automated chromatographic system. Proteins were eluted into four fractions (A, B, C, and D), in which A and B contained non-gelatin-binding proteins, and C and D fractions contained gelatin-binding proteins. Gels were analyzed by Quantity One software, in which five protein bands were detected in fraction D, with molecular weights between 12 and 30 kDa. The gelatin-binding proteins (fraction D) were loaded into a HiTrap™ Heparin HP affinity column. Two chromatographic fractions were separated (D1 and D2), in which D1 contained non-heparin-binding proteins, and D2 contained heparin-binding proteins. Proteins from the last two peaks were subjected to 12.5% SDS-PAGE and Western Blot. Two bands with molecular weight of 14 and 24 kDa, contained in fraction D1, were excised from gel and subjected to tandem mass spectrometry, identifying the proteins RSVP14 and RSVP24. Thus, the chromatographic methods of the present study are efficient to capture the enriched fraction of RSVP14. |
publishDate |
2020 |
dc.date.none.fl_str_mv |
2020-01-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-35982020000100407 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-35982020000100407 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.37496/rbz4920190166 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Sociedade Brasileira de Zootecnia |
publisher.none.fl_str_mv |
Sociedade Brasileira de Zootecnia |
dc.source.none.fl_str_mv |
Revista Brasileira de Zootecnia v.49 2020 reponame:Revista Brasileira de Zootecnia (Online) instname:Sociedade Brasileira de Zootecnia (SBZ) instacron:SBZ |
instname_str |
Sociedade Brasileira de Zootecnia (SBZ) |
instacron_str |
SBZ |
institution |
SBZ |
reponame_str |
Revista Brasileira de Zootecnia (Online) |
collection |
Revista Brasileira de Zootecnia (Online) |
repository.name.fl_str_mv |
Revista Brasileira de Zootecnia (Online) - Sociedade Brasileira de Zootecnia (SBZ) |
repository.mail.fl_str_mv |
||bz@sbz.org.br|| secretariarbz@sbz.org.br |
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1750318153674522624 |