Total-SYK assay principle

The Total SYK assay quantifies the expression level of SYK in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-SYK 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 SYK 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-SYK 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-SYK HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-SYK one-plate assay protocol

Detection of Total SYK 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.

SYK activation in anti-IgM-stimulated Ramos B cells

The human B cell lymphoma Ramos cell line was seeded in a half 96-well culture-treated plate at 100,000 cells/well in 20 µL FBS-free culture medium. After an overnight incubation at 37 °C, 5% CO2, cells were stimulated with 10 µL of increasing concentrations of an anti-human IgM antibody for 10 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-SYK (Tyr525/526) or Total-SYK detection reagents were added. The HTRF signal was recorded after an overnight incubation.

The anti-human IgM antibody induced a dose-dependent increase in SYK phosphorylation at Tyr525/526 without changing the expression level of the protein, demonstrating the activation of the BCR complex at the cell surface.

SYK inhibition in macrophage-differentiated THP-1 cells treated with P505-15

The human monocytic THP-1 cell line was seeded in a 96-well culture-treated plate at 200,000 cells/well and differentiated into macrophages by incubation with 100 ng/mL PMA for 24h at 37 °C, 5% CO2. After differentiation, cells were first treated with increasing doses of the SYK inhibitor P505-15 for 1h, and then with 250 µM pervanadate for 10 minutes. After treatment, cells were lyzed with 50 µL of supplemented lysis buffer #3 (1X) for 30 minutes at RT under gentle shaking, and 16 µL of cell lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Phospho-SYK (Tyr525/526) or Total-SYK detection reagents. The HTRF signal was recorded after an overnight incubation.

As expected, the SYK inhibitor P505-15 induced a dose-dependent decrease in SYK phosphorylation at Tyr525/526, while the expression level of the kinase was not modulated by the treatment.

HTRF total SYK 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 10 minutes, and after an additional centrifugation step, cells were lyzed with 10 mL of supplemented lysis buffer #3 (1X) 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-SYK detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF total-SYK assay, 500 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 total-SYK assay was 4 times more sensitive than the Western Blot technique.