PathHunter™ ß-arrestin Assays

PathHunter™ ß-arrestin Assays

DiscoveRx Corporation

PathHunter™ ß-arrestin Assays are revolutionary high-throughput screening assays for monitoring G-protein coupled receptor (GPCR) activation following ligand stimulation, without an imaging more

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PathHunter™ ß-arrestin Assays 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. The chemiluminescent signal generated can easily be read in 96-, 384- or 1536-well microplates with a standard luminometer, so library screening is simple, fast, and cost effective.

Activation Assay for Virtually Any GPCR
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.

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Features and Benefits
» Ideally suited for HTS with a single-addition, chemiluminescent detection protocol
» Directly screen broadest range of GPCRs— or deorphanize novel GPCRS
» Using a standard plate reader, the protocol is fast, simple, and reduces operating costs
» Use common cell types

PathHunter™ ß-arrestin Assay Principle
The assay detects binding of an agonist to a GPCR of interest by directly measuring ß-arrestin binding to the GPCR. Once bound, complementation occurs between the two ß-galactosidase components: the ProLink tag is fused to the C-terminus of the GPCR of interest; the Enzyme Acceptor (EA) is attached to ß-arrestin. These components interact only when in close proximity, forming active ß-gal enzyme that converts substrate to detectable signal.* 

Kit Contents
» ProLink™ Cloning Vector
» PathHunter™ ß-Arrestin CHO-K1, HEK 293, or U2OS Cell Lines
» PathHunter™ Detection Reagents

Instrument Requirements
» Luminometer, Multimode Microplate Reader or CCD Camera

* Covered by issued and pending US and foreign patents.


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