7TM Phosphorylation Assay Kits

Analysis of agonist-driven GPCR phosphorylation provides insights into the receptor activation state and ligand pharmacology. The 7TM Phosphorylation Assay is the first-in-class immunoassay for the quantitative assessment of GPCR phosphorylation that can be performed entirely in multiwell cell culture plates thus eliminating the need for Western blot analysis. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by detection using Premium Phosphosite-Specific 7TM Antibodies as phospho-biosensors (Figure 1). The 7TM Receptor Phosphorylation Assay allows quantitative determination of GPCR phosphorylation particular when total receptor is determined in parallel using Non-Phospho 7TM Antibodies, which are included in each kit. The 7TM Receptor Phosphorylation Assay can be performed using transiently or stably transfected cells. This assay protocol has been optimized for transiently transfected HEK293 cells in 96-well format using plasmids expressing 3xHA-tagged GPCR constructs. It can be adapted to other affinity tags such as FLAG, Myc, His or GFP. The assay can be performed manually or fully automated in medium- to high-throughput mode. The 7TM Phosphorylation Assay is fast, robust and reliable, thus fulfilling all the requirements for extensive application in academic and pharmaceutical research.

Figure 1. Step-by-step flowchart showing the 7TM phosphorylation assay protocol. (1) Cells expressing affinity-tagged GPCRs are grown in F-bottom cell culture plates, and upon reaching ≥ 95% confluency, the cells are exposed to the agonist, antagonist or inhibitor of interest. (2) The cells are lysed in detergent buffer, and lysates are cleared by centrifugation. (3) For parallel detection of phosphorylated and total receptors, the lysate of each sample is divided and transferred into two corresponding wells of U-bottom assay plates. (4) Anti-tag magnetic beads are added to each well for receptor immunoprecipitation. (5) Primary phosphosite-specific and phosphorylation-independent antibodies are added to the appropriate wells of each sample. (6) A secondary enzyme-labeled antibody is then added. (7) An enzyme substrate solution is added for detection, the color reaction is stopped by adding a stop solution. (8) Determine the optical density at 405 nm with a microplate reader. (9) Calculate concentration-response-curves. 

Figure 2. Schematic model of the recommended 7TM phosphorylation assay setup to determine concentration-response-curves of agonists or antagonists. Cells are grown in 96-F-bottom well culture plates (left panel). Cells are lysed and each lysate is divided and transferred into two corresponding wells of U-bottom assay plates (right panel). Thus, each well used for detection of phosphorylated receptor (phospho-site) has a corresponding well used for detection of total receptor as a loading control (non-phospho) (e.g. A1 and C1, etc.). Rows A to D show a typical example for agonist stimulation (green). Rows E to H show a typical example for antagonist challenge (red). Standard agonist stimulation is depicted in gray. Rows A and B as well as E and F are detected using phosphosite-specific antibodies. Rows C and D as well as G and H are used for detection of total receptors using phosphorylation-independent antibodies. Columns 1 and 2 are used for stimulation with standard agonist. Columns 3 to 9 are used for a 7-point concentration-response curve. If required columns 10 and 11 are available control stimulations. Column 12 serves as negative control in which no primary antibody is added resulting in background signal. Each stimulation is performed in duplicate.