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.

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.

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.

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.

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.

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.