Description
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 n 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. ons of AP2 beta ion cause specific dampening of cellular responses to stimuli such as hormones, neurotransmitters, or sensory signals. As AP2 essed in various tissues, it is believed that AP2 may play a major role in many cancers or other human diseases (diabetes) by ing receptor-mediated ns.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
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.
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.
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.
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.
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.
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.
