Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin
Autor(a) principal: | |
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Data de Publicação: | 2007 |
Outros Autores: | , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/1822/14035 |
Resumo: | The non-linearity of the fluorescence emission on increasing the probe to protein ratio has long been regarded as problematic and has lead to the development of dyes to overcome this effect. One of the causes of this non-linear response can be ascribed to the overlap of the label’s own absorption and emission spectra. At higher labelling ratios, this affords the possibility of a reasonably efficient energy migration pathway, thus reducing the observed quantum yield of the dye. In this work we study the photophysics of fluorescein isothiocyanate (FITC) when conjugated to bovine serum albumin (BSA) at different labelling ratios (in the range FITC: BSA 1 : 17–15 : 1) using both steady state and time-resolved fluorescence techniques where on going from under labelled to over labelled samples a decrease in the initial (and steady state) anisotropy is observed, accompanied by an increase in the complexity of the decay kinetics and a decrease in the average lifetime. The band structure, elucidated by synchronous scan fluorescence spectroscopy, is also found to change on increased labelling. These results can be applied to the study of protein conformation and were confirmed by the analysis of denaturing BSA using urea. |
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Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albuminScience & TechnologyThe non-linearity of the fluorescence emission on increasing the probe to protein ratio has long been regarded as problematic and has lead to the development of dyes to overcome this effect. One of the causes of this non-linear response can be ascribed to the overlap of the label’s own absorption and emission spectra. At higher labelling ratios, this affords the possibility of a reasonably efficient energy migration pathway, thus reducing the observed quantum yield of the dye. In this work we study the photophysics of fluorescein isothiocyanate (FITC) when conjugated to bovine serum albumin (BSA) at different labelling ratios (in the range FITC: BSA 1 : 17–15 : 1) using both steady state and time-resolved fluorescence techniques where on going from under labelled to over labelled samples a decrease in the initial (and steady state) anisotropy is observed, accompanied by an increase in the complexity of the decay kinetics and a decrease in the average lifetime. The band structure, elucidated by synchronous scan fluorescence spectroscopy, is also found to change on increased labelling. These results can be applied to the study of protein conformation and were confirmed by the analysis of denaturing BSA using urea.Royal Society of ChemistryUniversidade do MinhoHungerford, GrahamBenesch, JohanMano, J. F.Reis, R. L.20072007-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/14035eng1474-905X10.1039/b612870j17277838info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T12:26:38Zoai:repositorium.sdum.uminho.pt:1822/14035Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:21:06.042748Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin |
title |
Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin |
spellingShingle |
Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin Hungerford, Graham Science & Technology |
title_short |
Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin |
title_full |
Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin |
title_fullStr |
Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin |
title_full_unstemmed |
Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin |
title_sort |
Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin |
author |
Hungerford, Graham |
author_facet |
Hungerford, Graham Benesch, Johan Mano, J. F. Reis, R. L. |
author_role |
author |
author2 |
Benesch, Johan Mano, J. F. Reis, R. L. |
author2_role |
author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Hungerford, Graham Benesch, Johan Mano, J. F. Reis, R. L. |
dc.subject.por.fl_str_mv |
Science & Technology |
topic |
Science & Technology |
description |
The non-linearity of the fluorescence emission on increasing the probe to protein ratio has long been regarded as problematic and has lead to the development of dyes to overcome this effect. One of the causes of this non-linear response can be ascribed to the overlap of the label’s own absorption and emission spectra. At higher labelling ratios, this affords the possibility of a reasonably efficient energy migration pathway, thus reducing the observed quantum yield of the dye. In this work we study the photophysics of fluorescein isothiocyanate (FITC) when conjugated to bovine serum albumin (BSA) at different labelling ratios (in the range FITC: BSA 1 : 17–15 : 1) using both steady state and time-resolved fluorescence techniques where on going from under labelled to over labelled samples a decrease in the initial (and steady state) anisotropy is observed, accompanied by an increase in the complexity of the decay kinetics and a decrease in the average lifetime. The band structure, elucidated by synchronous scan fluorescence spectroscopy, is also found to change on increased labelling. These results can be applied to the study of protein conformation and were confirmed by the analysis of denaturing BSA using urea. |
publishDate |
2007 |
dc.date.none.fl_str_mv |
2007 2007-01-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/1822/14035 |
url |
http://hdl.handle.net/1822/14035 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
1474-905X 10.1039/b612870j 17277838 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Royal Society of Chemistry |
publisher.none.fl_str_mv |
Royal Society of Chemistry |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
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1799132676297326592 |