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Total Beta-arrestin 2 cellular kit HTRF®

The Total Beta-arrestin 2 kit enables the cell-based quantitative detection of beta-arrestin 2, for monitoring GPCR activity.
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  • Highly specific Highly specific
  • Ready-to-use Ready-to-use
  • No-wash No-wash
The Total Beta-arrestin 2 kit enables the cell-based quantitative detection of beta-arrestin 2, for monitoring GPCR activity.
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Overview

The Total B-arrestin 2 cellular assay monitors total B-arrestin 2, and is used to detect the expression of endogenous or overexpressed B-arrestin 2 in various cells. B-arrestin 2 are members of the arrestin/beta-arrestin protein family, and are known to be implicated in the agonist-mediated desensitization of G-protein-coupled receptors, and to cause specific dampening of cellular responses to stimuli such as hormones, neurotransmitters, or sensory signals. As B-arrestin 2 is expressed in various tissues, it is believed that B-arrestin 2 may play a major role in many cancers or other human diseases (diabetes...) by regulating receptor-mediated functions.

Total B-arrestin 2 assay principle

The Total B-arrestin 2 assay quantifies the expression level of B-arrestin 2 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses two labeled antibodies: One coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of B-arrestin 2 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Principle of the HTRF total B-arrestin 2 assay

Total B-arrestin 2 two-plate assay protocol

Detection of total B-arrestin 2 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Two-plate protocol of the HTRF total B-arrestin 2 assay

Total B-arrestin 2 one-plate assay protocol

Detection of total B-arrestin 2 with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

One-plate protocol of the HTRF total B-arrestin 2 assay

HTRF total B-arrestin 2 assay compared to Western Blot

HEK293 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After 72h incubation, the cells were lysed with 3 mL of lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using lysis buffer, and 13 µL of each dilution were transferred into a low volume white microplate before the addition of 3µL Lysis buffer + 4 µL of HTRF Total-B-arrestin 2 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

The side by side comparison of Western Blot and HTRF demonstrates that the HTRF assay is 4-fold more sensitive than the Western Blot, at least under these experimental conditions.

Comparison of HTRF total B-arrestin 2 kit with Western Blot

Species compatibility of HTRF total B-arrestin 2 assay

Human (HEK293), Hamster (CHO-K1), and murine (C2C12) cells were plated at 100,000 cells/ well in a 96 well plate in cell culture medium, and incubated for 24h at 37°C, 5% CO2. Medium was then removed and cells were lysed with 50 µL of lysis buffer for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF Total B-arrestin 2 detection reagents were added. The HTRF signal was recorded after 3h incubation at room temperature.

Expression levels correlate well with data from literature and with qPCR experiments performed with the same cells as the HTRF assays.

Illustrations with HTRF B-arrestin 2 kit on human, mouse and hamster cells

Simplified pathway of GPCR signaling and role of B-arrestins

B-arrestins play central roles in the GPCR signaling pathways by regulating agonist-mediated GPCR signaling. Among B-arrestin implications, they mediate both receptor desensitization and resensitization processes, act as a signaling scaffold for MAPK pathways, such as MAPK1/3 or AKT1, and participate in the recruitment of the ubiquitin-protein ligase to the receptors. Beta-arrestins function as multivalent adapter proteins that can switch the GPCR from a G-protein signaling mode (that transmits short-lived signals from the plasma membrane) to a beta-arrestin signaling mode that transmits a distinct set of signals initiated as the receptor internalizes and transits the intracellular compartment. 

During the GPCR desensitization process, B-arrestins bind to the GRK-phosphorylated receptor and sterically preclude its coupling to the G-protein.

B-arrestins target many receptors for internalization by acting as endocytic adapters, and recruit GPCRs into the adapter protein 2 complex 2 (AP-2) in clathrin-coated pits. Internalized arrestin-receptor complexes traffic to intracellular endosomes. Furthermore different modes of arrestin-mediated internalization occur, depending on the receptors and the cell types. 

Receptor resensitization requires receptor-bound arrestin to be removed, so that the receptor can be dephosphorylated and returned to the plasma membrane.

The extent of beta-arrestin involvement appears to vary significantly, depending on the receptor, the agonist, and the cell type.

GPCRs signaling pathway

GPCR signaling in cardiovascular research addressed with HTRF 2nd messenger assays

See how peer researchers monitor GPCR signaling with HTRF - Notes d'application

GTP binding assay : Gαi agonist and antagonist guides

Discover how to get the most out of your Gαi agonist and antagonist research with our HTRF GTP binding assays. - Guides

A new specific kit for characterizing compounds inducing B-arrestin2 recruitment at GPCR

Discover how the β-arr2 recruitment kit can help you study the interaction between AP-2 and β-arrestin2. - Notes d'application

GPCR compounds identification and pharmacological characterization with GTP Gi binding assay

Characterization of candidate molecules through GTP binding assays - Notes d'application

Highly specific tools for β arrestin monitoring in various cells

A convincing demonstration of monitoring β -arrestin 1 and 2 expressed in various cellular models - Notes d'application

How to run a β-arrestin2 assay | PerkinElmer

6' video to set up your β-arrestin2 assay - Vidéos

eBook | Exploring β-Arrestins and Their Therapeutic Potential

Deepen Your β-Arrestin Knowledge - Guides

GPCRs: the pathway to Discovery

Complete solutions for GPCR Drug Discovery - Brochures

Safety Data Sheet (DEU) B-arrestin 2 / 64BAR2TPEB

64BAR2TPEB - Fiches de sécurité

Safety Data Sheet (ELL) B-arrestin 2 / 64BAR2TPEB

64BAR2TPEB - Fiches de sécurité

Safety Data Sheet (ITA) B-arrestin 2 / 64BAR2TPEB

64BAR2TPEB - Fiches de sécurité

Safety Data Sheet (SPA) B-arrestin 2 / 64BAR2TPEB

64BAR2TPEB - Fiches de sécurité

Safety Data Sheet (DEU) B-arrestin 2 / 64BAR2TPEC

64BAR2TPEC - Fiches de sécurité

Safety Data Sheet (ELL) B-arrestin 2 / 64BAR2TPEC

64BAR2TPEC - Fiches de sécurité

Safety Data Sheet (ITA) B-arrestin 2 / 64BAR2TPEC

64BAR2TPEC - Fiches de sécurité

Safety Data Sheet (SPA) B-arrestin 2 / 64BAR2TPEC

64BAR2TPEC - Fiches de sécurité

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