Time-resolved fluorescence resonance energy transfer kinase assays using physiological protein substrates: Applications of terbium–fluorescein and terbium–green fluorescent protein fluorescence resonance energy transfer pairs

Fluorescence-based kinase assays using peptide substrates are an established format for high-throughput screening and profiling of kinases. Among fluorescence-based formats, time-resolved fluorescence resonance energy transfer (TR-FRET) using a lanthanide donor species has advantages over other fluorescent formats in being resistant to many types of optical interference such as autofluorescent compounds, scattered light from precipitated compounds, or colored compounds that absorb excitation or emission radiation (“color quenchers”). By taking advantage of the fact that acceptors such as fluorescein or green fluorescent protein (GFP) can be paired with a terbium donor in a TR-FRET assay, we have developed TR-FRET kinase assays that use physiologically relevant native protein substrates, either labeled with fluorescein or expressed as GFP fusions. Phosphorylation of the labeled protein substrate results in an increase in TR-FRET when incubated with a terbium-labeled antibody that specifically recognizes the phosphorylated product. Thus, a strategy of using terbium-based TR-FRET can be applied to develop kinase assays, and the unique properties of terbium lead to a high degree of flexibility with regard to specific assay design.