Phospho-PLCg2 (Tyr1217) assay principle

The Phospho-PLCg2 (Tyr1217) assay measures PLCg2 when phosphorylated at Tyr1217. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-PLCg2 (Tyr1217) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-PLCg2 (Tyr1217) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-PLCg2 (Tyr1217) one-plate assay protocol

Detection of Phosphorylated PLCg2 (Tyr1217) 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.

PLCg2 activation in anti-IgM-stimulated Ramos B cells

The human B cell lymphoma Ramos cell line was seeded in a half area 96-well culture-treated plate at 400,000 cells/well in 25 µL complete culture medium. After 3 hour incubation at 37 °C, 5% CO2, cells were stimulated with 5 µL of increasing concentrations of an anti-human IgM antibody for 5 minutes, and then lyzed with 10 µL of supplemented lysis buffer #4 (4X) for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-PLCg2 (Tyr1217) or Total-PLCg2 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

The anti-human IgM antibody induced a dose-dependent increase in PLCg2 phosphorylation at Tyr1217 without changing the expression level of the protein, demonstrating the activation of the BCR complex at the cell surface.

HTRF phospho-PLCg2 (Tyr1217) assay compared to western blot

The human B cell lymphoma Ramos cell line was cultured in a T175 flask in complete culture medium for 48h at 37 °C, 5% CO2. After centrifugation, pelleted cells were stimulated with 20 µg/mL of an anti-human IgM antibody for 5 minutes. Then lysis buffer #4 (2X) was added for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-PLCg2 (Tyr1217) detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF phospho-PLCg2 (Tyr1217) assay, 4,000 cells/well were enough to detect a significant signal, while 8,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF phospho-PLCg2 assay was 2 times more sensitive than the Western Blot technique.

Simplified pathway of PLCg2 signaling

PLCg2 (Phospholipase C gamma 2) is a cytoplasmic tyrosine kinase which plays a crucial role in signal transduction, especially in immune cells such as B cells and microglia (brain-resident macrophages).

Upon the stimulation of B cells or TREM2/DAP12 receptor complexes, PLCg2 interacts with SYK, BTK, and BLNK (also called SLP-65), leading to the activation by phosphorylation of its catalytic domain at Tyr1217. PLCg2 will then cleave the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The IP3 will then increase calcium release and degradate it into IP1.

This will trigger the activation of multiple signaling pathways and cellular responses, including cell activation, proliferation, and survival.