Fluorescence polarization discriminates green fluorescent protein from interfering autofluorescence in a microplate assay for genotoxicity.
An unconventional use for the polarization optics, associated with a variety of commercially available fluorescence microplate readers, is reported. This novel application has allowed the discrimination of green fluorescent protein (GFP) fluorescence in genetically modified yeast cells from interfering autofluorescent species. The method exploits the unusually high fluorescence anisotropy of GFP compared to smaller fluorophores and autofluorescent species. The principle was successfully applied to resolve the induced GFP signal from that of autofluorescent test compounds, in an assay for genotoxic species. The use of fluorescence polarization enabled both proflavin and methapyrilene to be identified as genotoxic agents in the yeast assay. This would not have been possible using conventional fluorescence alone since these compounds were found to be intensely autofluorescent at the same wavelength as GFP and thus effectively mask the GFP signal.
Knight
Andrew W
AW
Department of Instrumentation and Analytical Science, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester M60 1QD, UK.
Goddard
Nicholas J
NJ
Billinton
Nicholas
N
Cahill
Paul A
PA
Walmsley
Richard M
RM
D016880:Anisotropy; D001671:Biochemistry / Q000379:methods; D002455:Cell Division; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D005454:Fluorescence Polarization; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry* / Q000494:pharmacology; D008956:Models, Chemical; D013050:Spectrometry, Fluorescence / Q000379:methods*; D015003:Yeasts / Q000378:metabolism
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins