PathHunter™ and PathHunter™ Flash Detection Kits

The PathHunter™ and PathHunter™ Flash Detection Kits provide a simple, homogeneous gain-of-signal assay for measurement of enzyme fragment complementation (EFC) activity in live cells. PathHunter Detection Reagents are designed for use with PathHunter Cell Lines expressing both the complementing fragments of β-galactosidase (β-gal). All assays have been validated for use in both 96-well and 384-well microplate formats.

Product Features

•Homogeneous and flexible assay protocol
•Simple, gain-of-signal assay
•Robust, reproducible signal (Z' factors > 0.7)
•Minimal compound interference
•Run on any standard benchtop luminometer

For instrument compatibility, please click here.

Glow Detection

The PathHunter Detection Kit was designed to measure intracellular EFC activity within 2 hours after addition of detection reagents. This kit is ideal for use with any standard benchtop luminometer. For a list of compatible instruments, click here.

Flash Detection

The PathHunter Flash Detection Kit was designed to measure interacellular EFC activity in less than one minute after addition of detection reagents. As a result, receptor activation can be detected very rapidly with minimal disruption of cell biology. This kit is ideal for instruments with onboard fluid handling and rapid chemiluminescent data acquisition, such as Hamamatsu FDSS6000, LumiLux™ Cellular Screening Platform, FLIPRTETRA, etc.

Compatible PathHunter™ Cell-Based Assays

PathHunter cell lines are engineered to express the complementing fragments β-gal in different cellular compartments.

PathHunter™ β-Arrestin assays detect GPCR activation by measuring the interaction of β-Arrestin with activated GPCRs. A small, 42 amino acid enzyme fragment, called ProLink was appended to the C-terminus of the GPCR. Arrestin was fused to the larger enzyme fragment, termed EA (Enzyme Acceptor). Activation of the GPCR stimulates binding of arrestin and forces the complementation of the two enzyme fragments, resulting in formation of a functional β-gal enzyme capable of hydrolyzing substrate and generating a chemiluminescent signal.

In the PathHunter Nuclear Translocation Assays, EA is localized in the cell nucleus and the small complementing PK fragment is expressed as a fusion protein to a target of interest in the cytoplasm. When a signaling pathway is activated a cascade of events ensues, resulting in the translocation of the target protein to the nucleus and forced complementation of the two β-gal fragments and formation of a functional enzyme that hydrolyzes substrate and generation of a chemiluminescent signal.

PathHunter™ Cell-Based Kinase Assays monitor the interaction of wild type, full length receptor tyrosine kinase proteins with SH2 phosphotyrosine binding domains using EFC. The assay does not require antibodies or washes and can be easily adapted to a one-step HTS-friendly assay.

PathHunter™ Mitotic Index Assays monitor mitotic activity in live cells by measuring the complementation of nuclear and cytoplasmically localized β-gal fragments. During mitosis, the nuclear envelope degrades and allows the two β-gal fragments to complement in the cytoplasm, forming active enzyme and generation of chemiluminescent signal.

Available Collaterals

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