Total-PLCg2 assay principle

The Total PLCg2 assay quantifies the expression level of PLCg2 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-PLCg2 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 the presence of PLCg2 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-PLCg2 two-plate assay protocol

The two-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 Total-PLCg2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-PLCg2 one-plate assay protocol

Detection of Total PLCg2 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 of FBS-free culture medium. After a 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.

Cell density in Ramos and THP-1 cells

The human B cell lymphoma Ramos and monocyte THP-1 cell lines were seeded in a half area 96-well culture-treated plate in 30 µL complete culture medium. After a 3 hour incubation at 37 °C, 5% CO2, cells were 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.

Expression of total PLCg2 is twice as high in Ramos cells as in THP-1.

HTRF total PLCg2 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 total PLCg2 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF total PLCg2 assay, 1,000 cells/well were enough to detect a significant signal, while 2,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF phospho-PLCg2 assay was twice as sensitive as the Western Blot technique.