Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available f...

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Autores principales:	Yoko Hayashi-Takanaka, Timothy J Stasevich, Hitoshi Kurumizaka, Naohito Nozaki, Hiroshi Kimura
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/15e68a6cd9e7421dbf4057d4259a37a7
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spelling	oai:doaj.org-article:15e68a6cd9e7421dbf4057d4259a37a72021-11-25T06:02:04ZEvaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.1932-620310.1371/journal.pone.0106271https://doaj.org/article/15e68a6cd9e7421dbf4057d4259a37a72014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25184362/?tool=EBIhttps://doaj.org/toc/1932-6203To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).Yoko Hayashi-TakanakaTimothy J StasevichHitoshi KurumizakaNaohito NozakiHiroshi KimuraPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 9, p e106271 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yoko Hayashi-Takanaka Timothy J Stasevich Hitoshi Kurumizaka Naohito Nozaki Hiroshi Kimura Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.
description	To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).
format	article
author	Yoko Hayashi-Takanaka Timothy J Stasevich Hitoshi Kurumizaka Naohito Nozaki Hiroshi Kimura
author_facet	Yoko Hayashi-Takanaka Timothy J Stasevich Hitoshi Kurumizaka Naohito Nozaki Hiroshi Kimura
author_sort	Yoko Hayashi-Takanaka
title	Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.
title_short	Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.
title_full	Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.
title_fullStr	Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.
title_full_unstemmed	Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.
title_sort	evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/15e68a6cd9e7421dbf4057d4259a37a7
work_keys_str_mv	AT yokohayashitakanaka evaluationofchemicalfluorescentdyesasaproteinconjugationpartnerforlivecellimaging AT timothyjstasevich evaluationofchemicalfluorescentdyesasaproteinconjugationpartnerforlivecellimaging AT hitoshikurumizaka evaluationofchemicalfluorescentdyesasaproteinconjugationpartnerforlivecellimaging AT naohitonozaki evaluationofchemicalfluorescentdyesasaproteinconjugationpartnerforlivecellimaging AT hiroshikimura evaluationofchemicalfluorescentdyesasaproteinconjugationpartnerforlivecellimaging
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Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

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