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HTRF Human and Mouse Total β-arrestin 1 Detection Kit, 1,000 Assay Points

The total Beta-arrestin 1 kit enables the cell-based quantitative detection of beta-arrestin 1, for monitoring GPCR activity.

For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Product Variants
Part number: 64BAR1TPEB
Unit Size: 1,000 Assay Points
List price: USD 3,160.00
Your price:
USD 3,160.00
USD 3,160.00 /each
Part number: 64BAR1TPEC
Unit Size: 20,000 Assay Points
List price: USD 20,360.00
Your price:
USD 0.00
USD 20,360.00 /each

Overview

The Total B-arrestin 1 cellular assay monitors total B-arrestin 1, and is used to detect the expression of endogenous or overexpressed B-arrestin 1 in various cells. The B-arrestin 1 are of the n/beta-arrestin protein family, and wn to be implicated in the -mediated desensitization of G-protein-coupled receptors. They also cause specific dampening of cellular responses to stimuli such as hormones, neurotransmitters, or sensory signals. As B-arrestin 1 is expressed in various tissues, it is believed that B-arrestin 1 may play a major role in many cancers or other human diseases (diabetes) by ing receptor-mediated ns.

Specifications

Assay Points
1000
Assay Target Type
Kit
Assay Technology
HTRF
Brand
HTRF
Quantity
1
Therapeutic Area
Cardiovascular
Inflammation
Metabolism/Diabetes
NASH/Fibrosis
Unit Size
1,000 Assay Points

Video gallery

How it works

Total B-arrestin 1 assay principle

The Total B-arrestin 1 assay quantifies the expression level of B-arrestin 1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total B-arrestin 1 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 1 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.

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Total B-arrestin 1 two-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of the Total B-arrestin 1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF total BRD2 assay

 

Total B-arrestin 1 one-plate assay protocol

Detection of total B-arrestin 1 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 BRD2 assay

 

Assay validation

HTRF total B-arrestin 1 assay compared to Western Blot

HepG2 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 1 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 16-fold more sensitive than the Western Blot, at least under these experimental conditions.

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Species compatibility of HTRF total B-arrestin 1 assay

Human (HepG2), Hamster (CHO-K1), and murine (NIH 3T3) 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 was transferred into a 384-well sv white microplate and 4 µL of the HTRF Total B-arrestin 1 detection reagents were added. The HTRF signal was recorded after 3h incubation at room temperature.

Expression levels are well correlated with data from literature and Western Blot experiments performed with the same cells as the HTRF assays.

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Simplified pathway

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 that are 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.

Then B-arrestins target many receptors for internalization by acting as endocytic adapters, and recruiting the GPCRs to 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 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.

4phospho-pathway-b-arrestin-total-1-64bar1tpeb-64bar1tpec-64bar1tpebj.svg

 

Resources

1-4 of 4 Resources
Application Note Icon Image
Application Note
Characterizing compounds acting on β-arrestin2 coupled GPCRs

Dive deeper into research on the GPCR signaling pathway

β-arrestins are intracellular proteins that play an important role in GPCR...

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Application Note
Highly specific tools for β-arrestin monitoring in various cells

<p>Improve your research on β-arrestin with HTRF™ technology</p><p>The β-arrestins 1 and 2 play a central role in GPCR signaling pathways by...</p>

Guide
HTRF solutions, guide to major applications

This guide provides you an overview of HTRF applications in several therapeutic areas.

Application Note Icon Image
Application Note
Optimization and pharmacological validation of wild type beta-arrestin 2 plasmid transfection.

In this note, we cover the best practices and elements that can be monitored and tested to achieve an effective transfection with...

SDS, COAs, Manuals and more Illuminator

SDS, COAs, Manuals and more

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