These assays are based upon the use of a europium or terbium chelate (a transition metal-ligand complex displaying long-lived fluorescent properties) and labeled anti-phosphopeptide or anti-phos-photyrosine antibodies that can bind to phosphorylated peptides. The antibodies are usually labeled with aromatic fluorescent tags such as Cy5, rhodamine, or fluorescent proteins such as allophyco-cyanin—a light-harvesting protein that absorbs at 650 nm and emits at 660 nm (Moshinsky et al., 2003; Newman and Josiah, 2004; Schroeter et al., 2008). When the anti-phosphotyrosine or anti-phosphopeptide antibodies bind to a labeled phosphorylated peptide, the proximity of the antibody to the labeled peptide results in a transfer of energy.
The energy transfer is a consequence of the emission spectrum of the metal-ligand complex overlapping with the absorption of the labeled peptide. If the donor fluor is within 7 to 9 nm of the acceptor fluor, Förster resonance energy transfer can occur although this distance can vary considerably (Vogel and Vedvik, 2006). The action of a kinase enzyme would increase the concentration of phosphopeptide over time and result in an increased signal in such an assay. The action of a phosphatase would decrease the signal in this assay format.
TR-FRET assays have two main advantages. The first is the time-gated signal detection; the emission is not measured for 100 to 900 ps after the initial excitation frequency is applied, resulting in a reduction in fluorescence background from the MTP's buffers and compounds. Data is acquired by multiple flashes per read, to help improve the sensitivity and reproducibility of the signal. The second advantage is that one can measure the ratio of the emission from the acceptor molecule to the emission from the donor molecule. Because of this "ratiometric" calculation, variations in signal due to variations in pipetting volume can be reduced. Generally one observes less inter-well variation with TR-FRET assays (Glickman et al., 2002).
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