title: Monitoring labeling reactions using fluorescence polarization.
abstract: Fluorescence techniques are widely applied in protein research owing to their specificity and sensitivity, but require prior fluorescent labeling. Here we show a novel approach to optimize labeling protocols by monitoring labeling reactions using fluorescence polarization: the larger molecular mass of the fluorescent protein conjugate compared to the dye alone results in an increase in fluorescence anisotropy during the reaction. Thereby, labeling of lysozyme with fluorescein isothiocyanate or carboxyfluorescein succinimidyl ester could be monitored and the influence of parameters such as the pH could be quantitatively assessed. Moreover, the impact and kinetics of side reactions such as hydrolysis were determined. This new method is rapid, easy to implement, and generically applicable.
authors.affiliation: Institute of Life Technologies, University of Applied Sciences and Arts Western Switzerland Valais, Route du Rawyl 64, CH-1950 Sion, Switzerland.
authors.forename: Yvan
authors.initials: Y
authors.lastname: Crittin
authors.affiliation: Institute of Life Technologies, University of Applied Sciences and Arts Western Switzerland Valais, Route du Rawyl 64, CH-1950, Switzerland Sion.
authors.forename: Julien
authors.initials: J
authors.lastname: Héritier
authors.affiliation: Institute of Life Technologies, University of Applied Sciences and Arts Western Switzerland Valais, Route du Rawyl 64, CH-1950 Sion, Switzerland. jmanuel.segura@hevs.ch.
authors.forename: Jean-Manuel
authors.initials: JM
authors.lastname: Segura
mesh_terms: D016880:Anisotropy; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D009113:Muramidase / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D013388:Succinimides / Q000737:chemistry; D013997:Time Factors; D014867:Water / Q000737:chemistry
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides; D014867:Water; D009113:Muramidase; D016650:Fluorescein-5-isothiocyanate