Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 361221
Title Sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye Nile re
Author(s) Sutter, M.; Oliveira, S.; Sanders, N.N.; Lucas, B.; Hoek, A. van; Hink, M.A.; Visser, A.J.W.G.; Smedt, S.C. de; Hennink, W.E.; Jiskoot, W.
Source Journal of Fluorescence 17 (2007)2. - ISSN 1053-0509 - p. 181 - 192.
DOI https://doi.org/10.1007/s10895-007-0156-6
Department(s) Biophysics
Biochemistry
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2007
Keyword(s) galactosidase escherichia-coli - beta-galactosidase - ftir spectroscopy - congo red - immunogenicity - surfaces - probe
Abstract The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein ß-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of ß-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 ß-galactosidase aggregates led to a shift of the emission maximum (¿ 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 ß-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native ß-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 ß-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.
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