Receptor Binding Assays using Fluorescence Polarization Detection: the [FP]2TM Characterized Tracers ( p. The only insurmountable l...)

Receptor Binding Assays using Fluorescence Polarization Detection: the [FP]2TM Characterized Tracers

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The only insurmountable limitation to FP with the current level of HTS reader sensitivity, available fluorophores and labeling capabilities is that primarily, only peptidic GPCR ligands are suitable. Small molecule ligands below 1000 amu are difficult to label while preserving biological activity. Ligands greater than 5000 Da are not only difficult to label, but their size tends to preclude adequate assay performance. While size-independent detection is available with other non-radiometric detection technologies (FMAT, FIDA), they are still limited from a fluorescence labeling perspective. Furthermore, each of these detection technologies requires a dedicated reader only available from the detection technology provider. In addition, each of these technologies relies on scanning the contents of the well, which typically requires read times/well greater than one (1) second and thus precludes ultra high throughput operation at this time.

This application note serves to demonstrate the broad applicability of fluorescence polarization detection for peptidic ligand binding to GPCR targets. In addition to the eight ligands described here, fully optimized kits (pharmacology, assay performance, DMSO tolerance, kinetics and stability) are available. See [FP]² brochure, reference number H78392, for more detailed lists.

Principles of Fluorescence Polarization Detection

The physical process that allows fluorescence polarization detection is centered on the principle that smaller molecules rotate faster than larger molecules in solution. Fluorescence can be used to probe these differences in rotation rates since the process of fluorescence, the time required for a fluorophore to emit a photon after excitation, also called the fluorescence lifetime, requires a measurable amount of time, typically in the nanosecond range for most fluorophores. If we assume that this time is typically a cons
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( p. The only insurmountable l...)Receptor Binding Assays using Fluorescence Polarization Detection: the [FP]2TM Characterized Tracers