FRET (Fluorescence Resonance Energy Transfer)

Principle: FRET is a proximity-based assay that detects energy transfer between a donor fluorophore and an acceptor when they are within 1–10 nm. The energy transfer occurs when the two labeled proteins come into close proximity, generating a detectable fluorescent signal.

Cellular Applications: FRET is widely used in cell-based studies to observe protein-protein interactions (PPIs), conformational changes, and signaling events in real time. It’s ideal for monitoring dynamic cellular processes and the spatial distribution of molecules.

TR-FRET (Time-Resolved FRET)

Advancement of FRET: TR-FRET improves upon traditional FRET by introducing a lanthanide-based donor with a long fluorescence lifetime (e.g., europium or terbium), which significantly reduces background noise from non-specific short-lived fluorescence in cells.

Principle: After a time delay following excitation, the fluorescence from the donor and acceptor is measured, allowing more sensitive detection in cell-based assays.

Applications in Cells: TR-FRET is particularly effective in high-throughput screening (HTS) in drug discovery, especially in detecting GPCR signaling or kinase interactions where the noise in standard FRET assays would obscure weak signals.

HTRF (Homogeneous Time-Resolved Fluorescence)

Refinement of TR-FRET: HTRF is a commercialized version of TR-FRET, combining the benefits of time-resolved fluorescence and ratiometric detection to provide highly robust and reliable measurements in cell-based assays.

Principle: In HTRF, donor molecules (typically europium cryptate) and acceptor molecules (often XL665) generate a measurable ratiometric signal that is directly proportional to the molecular interaction within the cell.

Applications: HTRF is highly popular for drug discovery, particularly in receptor-ligand binding assays, protein interaction studies, and intracellular signaling detection. Its robustness makes it especially suitable for large-scale cell-based assays and quantitative analysis of PPIs.

▶ GPCR HTRF Assays

HTRF cAMP Assay: This assay measures intracellular cAMP levels, a key second messenger in GPCR signaling, commonly used for Gαs and Gαi-coupled receptors. It quantifies the receptor's response to activation, useful for agonist/antagonist screening in live cells.

HTRF IP1 Assay: This assay detects IP1 accumulation, reflecting Gq-coupled GPCR activity. It is highly effective for studying Gq-mediated signal transduction and GPCR ligand characterization.

HTRF Tag-Lite Binding Assay: This binding assay measures real-time ligand-GPCR interactions by tagging GPCRs and detecting ligand binding via FRET, ideal for ligand screening and kinetic studies.