Phospho-PLCG1 (tyr783) assay principle

The Phospho-PLCg1 (Tyr783) assay measures PLCg1 when phosphorylated at Tyr783. 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-PLCg1 (Tyr783) 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-PLCg1 (Tyr783) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-PLCg1 (Tyr783) one-plate assay protocol

Detection of Phosphorylated PLCg1 (Tyr783) 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.

PLCg1 activation in anti-CD3-stimulated Jurkat T cells

The human Jurkat T cell line was seeded in a half area 96-well culture-treated plate at 200,000 cells/well in 25 µL complete culture medium. After a 3 hour incubation at 37 °C, 5% CO2, cells were stimulated with 5 µL of increasing concentrations of an CD3 antibody for 2 minutes, and then lyzed with 10 µL of supplemented lysis buffer #3 (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-PLCg1 (Tyr783) or Total-PLCg1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

The anti-CD3 antibody induced a dose-dependent increase in PLCg1 phosphorylation at Tyr783 without changing the expression level of the protein, demonstrating the activation of the TCR complex at the cell surface.

 PLCg1 activation in EGF-stimulated A431 cells

The human A431 cell line was seeded in a 96-well culture-treated plate at 50,000 cells/well in 50 µL complete culture medium. After overnight incubation at 37 °C, 5% CO2, cells were stimulated with 50 µL of increasing concentrations of EGF for 5 minutes,

After 30 minutes of lysis incubation, phosphorylated and total PLCg1 were measured using the two-plate assay protocol. The HTRF signal was recorded after an overnight incubation.

The EGF induced a dose-dependent increase in PLCg1 phosphorylation at Tyr783 without changing the expression level of the protein, demonstrating the activation of tyrosine kinase receptor EGFR at the cell surface.

PLCg1 activation in PDGF-BB stimulated NIH-3T3 cells

The mouse NIH-3T3 cell line was seeded in a 96-well culture-treated plate at 100,000 cells/well in 50 µL complete culture medium. After overnight incubation at 37 °C, 5% CO2, cells were stimulated with 50 µL of increasing concentrations of PDGFR-BB for 30 minutes.

After 30 minutes of lysis incubation, phosphorylated and Total PLCg1 were measured using the two-plate assay protocol. The HTRF signal was recorded after an overnight incubation.

The NIH-3T3 induced a dose-dependent increase in PLCg1 phosphorylation at Tyr783 without changing the expression level of the protein, demonstrating the activation of tyrosine kinase receptor PDGFR at the cell surface.

HTRF phospho-PLCg1 (Tyr783) assay compared to Western Blot

The human Jurkat T 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 5 µg/mL of an anti-CD3 antibody for 2 minutes. Then lysis buffer #3 (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-PLCg1 (Tyr783) detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF phospho-PLCg1 (Tyr783) assay, 500 cells/well were enough to detect a significant signal, while 4,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF phospho-PLCg1 assay was 8 times more sensitive than the Western Blot technique.