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Total AP2 cellular kit HTRF®

The total AP2 kit enables the cell-based quantitative detection of AP2, for monitoring GPCR activity.

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  • Ready-to-use Ready-to-use
  • No-wash No-wash

The total AP2 kit enables the cell-based quantitative detection of AP2, for monitoring GPCR activity.

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Overview

The Total AP2 cellular assay monitors total AP2 beta, and is used to detect the expression of endogenous or overexpressed AP2 beta in various cells. AP2 beta are components of the adaptor protein complex 2 (AP-2) and are known to function as cargo proteins. They play several roles in cells, including the internalization of GPCRs through a clathrin-dependent endocytosis process by direct interaction with beta arrestins.  Variations of AP2 beta expression cause specific dampening of cellular responses to stimuli such as hormones, neurotransmitters, or sensory signals. As AP2 is expressed in various tissues, it is believed that AP2 may play a major role in many cancers or other human diseases (diabetes...) by regulating receptor-mediated functions.

Total AP2 assay principle

The Total AP2 assay quantifies the expression level of AP2 beta in a cell lysate. Unlike Western Blot, the assay is entirely plate-based, and does not require gels, electrophoresis, or transfer. The Total AP2 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of AP2 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 AP2 assay

Total AP2 two-plate assay protocol

Detection of total AP2 ​​​​​​​ 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 AP2 assay

Total AP2 one-plate assay protocol

Detection of total AP2 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 AP2 assay

HTRF total AP2 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-AP2 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.

Comparison of HTRF total AP2 kit with Western Blot

Species compatibility of HTRF total AP2 assay

Human (HEK293), Hamster (CHO-K1), and murine (NIH3T3) 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 AP2 detection reagents were added. The HTRF signal was recorded after 3h incubation at room temperature.

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

Illustrations with HTRF AP2 kit on human, mouse and hamster cells

Simplified pathway of GPCR signaling and role of AP2

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 in clathrin-coated pits by interacting with the β-adaptin subunit of the AP2 complex, and so recruit the GPCRs into a multipartite complex. The interaction between B-arrestin and AP2 is the central initial step in localizing the GPCRs into the clathrin-coated pits following agonist stimulation, and so pharmacological compounds targeting this interaction are of considerable interest. 


Internalized arrestin-receptor complexes traffic to intracellular endosomes. 

Receptor resensitization then 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, agonist, and cell type.



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Plate Reader Requirement

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