Use of fluorescence lifetime technology to provide efficient protection from false hits in screening applications.
This article describes novel data analysis of fluorescence lifetime-based protein kinase assays to identify and correct for compound interference in several practical cases. This ability, together with inherent advantages of fluorescence lifetime technology (FLT) as a homogeneous, antibody-free format independent of sample concentration, volume, excitation intensity, and geometry, makes fluorescence lifetime a practical alternative to the established "gold standards" of radiometric and mobility shift (Caliper) assays. The analysis is based on a photochemical model that sets constraints on the values of fluorescence lifetimes in the time responses of the assay. The addition of an exponential component with free floating lifetime to the constrained model, in which the lifetimes are constants predetermined from control measurements and the preexponential coefficients are "floating" parameters, allows the relative concentration of phosphorylated and nonphosphorylated substrates to be calculated even in the presence of compound fluorescence. The method is exemplified using both simulated data and experimental results measured from mixtures of dye-labeled phosphorylated and nonphosphorylated kinase substrates. A change of the fluorescence lifetime is achieved by the phosphorylated substrate-specific interaction with a bifunctional ligand, where one binding site interacts with the phosphate group and the other interacts with the dye.
Gakamsky
Dmitry M
DM
Edinburgh Instruments, Kirkton Campus, Livingston, West Lothian EH54 7DQ, UK. dgakamsky@edinst.com
Dennis
Richard B
RB
Smith
S Desmond
SD
D000585:Aminacrine / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D057075:Enzyme Assays / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D007501:Iron / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis*
D005456:Fluorescent Dyes; D000585:Aminacrine; D007501:Iron; D011494:Protein Kinases