Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red
The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 5...
Autores principales: | , , , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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Kluwer Academic Publishers-Plenum Publishers
2007
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1915606/ https://www.ncbi.nlm.nih.gov/pubmed/17294134 http://dx.doi.org/10.1007/s10895-007-0156-6 |
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author | Sutter, Marc Oliveira, Sabrina Sanders, Niek N. Lucas, Bart van Hoek, Arie Hink, Mark A. Visser, Antonie J. W. G. De Smedt, Stefaan C. Hennink, Wim E. Jiskoot, Wim |
author_facet | Sutter, Marc Oliveira, Sabrina Sanders, Niek N. Lucas, Bart van Hoek, Arie Hink, Mark A. Visser, Antonie J. W. G. De Smedt, Stefaan C. Hennink, Wim E. Jiskoot, Wim |
author_sort | Sutter, Marc |
collection | PubMed |
description | The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λ(max)) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages. |
format | Text |
id | pubmed-1915606 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | Kluwer Academic Publishers-Plenum Publishers |
record_format | MEDLINE/PubMed |
spelling | pubmed-19156062007-07-13 Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red Sutter, Marc Oliveira, Sabrina Sanders, Niek N. Lucas, Bart van Hoek, Arie Hink, Mark A. Visser, Antonie J. W. G. De Smedt, Stefaan C. Hennink, Wim E. Jiskoot, Wim J Fluoresc Original Paper The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λ(max)) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages. Kluwer Academic Publishers-Plenum Publishers 2007-02-09 2007-03 /pmc/articles/PMC1915606/ /pubmed/17294134 http://dx.doi.org/10.1007/s10895-007-0156-6 Text en © Springer Science+Business Media, LLC 2007 |
spellingShingle | Original Paper Sutter, Marc Oliveira, Sabrina Sanders, Niek N. Lucas, Bart van Hoek, Arie Hink, Mark A. Visser, Antonie J. W. G. De Smedt, Stefaan C. Hennink, Wim E. Jiskoot, Wim Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red |
title | Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red |
title_full | Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red |
title_fullStr | Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red |
title_full_unstemmed | Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red |
title_short | Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red |
title_sort | sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye nile red |
topic | Original Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1915606/ https://www.ncbi.nlm.nih.gov/pubmed/17294134 http://dx.doi.org/10.1007/s10895-007-0156-6 |
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