Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Antagonist activity at recombinant human TAS2R14 transiently expressed in HEK293T co-expressing Galpha16gust44 assessed as inhibition of aristolochic acid-induced intracellular calcium level by measuring remaining activity at 25 uM by fluo-4AM dye based FLIPR assay relative to control
Agonist activity at recombinant human N-terminal rat ST3 receptor-fused TAS2R14 expressed in HEK293T cells co-expressing Galpha16gust44 assessed as increase in intracellular calcium level at 100 uM by FLIPR assay
Antagonist activity at recombinant human N-terminal rat ST3 receptor-fused TAS2R14 expressed in HEK293T cells co-expressing Galpha16gust44 assessed as inhibition of aristolochic acid-induced increase in intracellular calcium level at 100 uM by FLIPR assay
Activity at human TAS2R14 expressed in HEK-293T-Galpha16-gustducin44 cells assessed as concentration of compound which reacts with receptor by measuring calcium response by FLIPR assay
Agonist activity at human TAS2R14 expressed in HEK293T cells co-expressing Galpha15 assessed as increase in intracellular calcium level by Calcium-3 dye based fluorescence assay
Agonist activity at human TAS2R14 expressed in HEK293T cells co-expressing Galpha15 assessed as increase in intracellular calcium level at 20 mM by Calcium-3 dye based fluorescence assay relative to control
Agonist activity at recombinant human N-terminal rat ST3 receptor-fused TAS2R14 expressed in HEK293T cells co-expressing Galpha16gust44 assessed as effect on intracellular calcium level at 100 uM by FLIPR assay
Activity at recombinant human N-terminal rat ST3 receptor-fused TAS2R14 expressed in HEK293T cells co-expressing Galpha16gust44 assessed as effect on aristolochic acids-induced intracellular calcium level at 100 uM by FLIPR assay
Agonist activity at human TAS2R14 transfected in HEK293T cells assessed as IP1 accumulation measured after 150 mins by IP1 accumulation assay relative to flufenamic acid
Agonist activity at human TAS2R14 transfected in HEK293T cells assessed as IP1 accumulation at 30 uM measured after 150 mins by IP1 accumulation assay relative to flufenamic acid
Agonist activity at human TAS2R14 transfected in HEK293T cells assessed as IP1 accumulation at 10 uM measured after 150 mins by IP1 accumulation assay relative to flufenamic acid
Partial agonist activity at human TAS2R14 transfected in HEK293T cells assessed as IP1 accumulation measured after 150 mins by IP1 accumulation assay relative to control
Potency index, ratio of flufenamic acid EC50 to test compound EC50 for human TAS2R14 transfected in HEK293T cells assessed as IP1 accumulation measured