title: Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation.
abstract: Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z' factors exceeding 0.6 are realised for this FLIM FRET assay.
authors.affiliation: Institute of Chemical Biology, Department of Chemistry, Imperial College London, South Kensington Campus, London, UK.
authors.forename: Dominic
authors.initials: D
authors.lastname: Alibhai
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Kelly
authors.forename: Sean
authors.initials: S
authors.lastname: Warren
authors.forename: Sunil
authors.initials: S
authors.lastname: Kumar
authors.forename: Anca
authors.initials: A
authors.lastname: Margineau
authors.forename: Remigiusz A
authors.initials: RA
authors.lastname: Serwa
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Thinon
authors.forename: Yuriy
authors.initials: Y
authors.lastname: Alexandrov
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Murray
authors.forename: Frank
authors.initials: F
authors.lastname: Stuhmeier
authors.forename: Edward W
authors.initials: EW
authors.lastname: Tate
authors.forename: Mark A A
authors.initials: MA
authors.lastname: Neil
authors.forename: Chris
authors.initials: C
authors.lastname: Dunsby
authors.forename: Paul M W
authors.initials: PM
authors.lastname: French
mesh_terms: D001331:Automation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D057054:Molecular Imaging / Q000379:methods*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry*
chemical_list: D054301:gag Gene Products, Human Immunodeficiency Virus