DiscoveRx PathHunter™ eXpress ß-Arrestin Assay for the Caliper LabChip®
DiscoveRx PathHunter™ ß-Arrestin GPCR Assay Description
PathHunter™ ß-Arrestin Assays developed by DiscoveRx are revolutionary high-throughput screening assays for monitoring G-protein coupled receptor (GPCR) activation following ligand stimulation, without an imaging instrument, fluorescent protein tag, or radioactivity. Instead, the assays detect GPCR activation through ß-arrestin binding to the expressed GPCR of interest, measuring the interaction of the two proteins using enzyme fragment complementation (EFC)*. The EFC approach offers a range of benefits for screening, including signal amplification, high assay windows, robust performance, and a homogenous, single-addition assay format.
Activation Assay for Broad Range of GPCR Targets
A wide variety of assays are available for analyzing GPCR signaling events, many of them based on second messenger detection. PathHunter ß-Arrestin Assays offer a generic method that can be applied to any GPCR and is based upon the interaction of arrestin with the GPCR during activation. The PathHunter ß-Arrestin Assay is unique compared to other arrestin assays in that it provides a direct measure of ß-Arrestin binding to the GPCR of interest, unlike imaging assays that detect movement of the arrestin molecule. Thus, the assays are ideal for HTS and may be very useful for deorphanizing novel GPCRs.
PathHunter™ ß-Arrestin Assay Principle
DiscoveRx PathHunter β-arrestin products monitor GPCR activity by detecting the interaction of arrestin with the activated GPCR using enzyme fragment complementation. In this system, the β-galactosidase enzyme is split into two inactive fragments. When these fragments are fused to two proteins that interact, the interaction of the two proteins forces the complementation of the fragments to restore enzyme activity. The figure below illustrates the system as applied to measuring GPCR activation. The larger portion of β-gal, termed EA for enzyme acceptor, is fused to the C-terminus of β-arrestin. The smaller, 4 kDa complementing fragment of β-gal, the ProLinkTM tag, is expressed as a fusion protein with the GPCR of interest at the C-terminus. Upon activation, the GPCR is bound by β-arrestin. The interaction of β-arrestin and the GPCR forces the interaction of ProLink and EA, thus allowing complementation of the two fragments of β-gal and the formation of a functional enzyme capable of hydrolyzing substrate and generating a fluorescence signal.
PathHunter eXpress ß-Arrestin GPCR Assay Description
With PathHunter eXpress Ready to assay GPCR kits, you can measure one or multiple GPCR responses to compound challenge with out having to invest in clonal cell lines. PathHunter eXpress kits are ideally suited for small to midsize screening and research laboratories, the kits come ready to use, include all the necessary reagents, media and plates and do not require expensive instrumentation to run. You can measure as few as one or as many* receptors needed to complete your screening or profiling experiments.
*Assay kits are available in single target and four target kits.
DiscoveRx eXpress kits are subject to the same development, validation, manufacturing, and quality control standards as our PathHunter clonal GPCR cell lines. The result is the most accurate, sensitive, and reproducible Cell based assay. Each receptor is evaluated and validated on the following performance: comparison to PathHunter Cell Lines, assay window, sensitivity, EC50 value compared to published data. Furthermore, Caliper compatible PathHunter eXpress cell lines were functionally tested on the Caliper LabChip® EZReader.
Intended Use
PathHunter eXpress kits offer a complete solution for your GPCR experiments. The eXpress kits contain everything needed (cells, detection reagents, media and plates) for a complete GPCR experiment*. PathHunter eXpress cells are quality tested and ready-to-screen; saving time and adding convenience. The assays have been designed for 96-well plate analyses. Cells have been manufactured for short term use in monitoring G-protein Coupled Receptor (GPCR) activation and arrestin recruitment.
* Test compounds are not included and must be provided by the researcher.
List of PathHunter eXpress ß-Arrestin GPCR Assays
