Phospho-SYK (Tyr525/526) assay principle

The Phospho-SYK (Tyr525/526) assay measures SYK when phosphorylated at Tyr525/526. 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 is 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-SYK (Tyr525/526) 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 Phospho-SYK (Tyr525/526) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-SYK (Tyr525/526) one-plate assay protocol

Detection of Phosphorylated SYK (Tyr525/526) 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.

TREM2 pathway activation in iPSC-macrophages

Parental (BIONi010-C, EBiSC #66540023) and TREM2 KO (BIONi010-C-17, EBiSC #66540632) human iPSC-derived macrophages, differentiated as described in Van Wilgenburg et al., 2013 (1), were plated in a 96-well plate at 40,000 cells/well. After 8 days of culture, cells were treated for 5 minutes with 5 µg/mL of an anti-TREM2 antibody (known to activate the receptor) or with the corresponding isotype control (Goat IgG antibody). After medium removal, 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 then transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Phospho-SYK (Tyr525/526) detection reagents. The HTRF signal was recorded after an overnight incubation.

As expected, the anti-TREM2 antibody induced an activation of the TREM2 pathway in the parental iPSC-macrophages, as highlighted by the increase in SYK phosphorylation at Tyr525/526, while the treatment did not modulate the phosphorylation status of the protein in the TREM2 KO cell line*.

*These data were obtained in collaboration with the Alzheimer’s Research UK Oxford Drug Discovery Institute (ODDI).

(1) Van Wilgenburg et al., Efficient, long term production of monocyte derived macrophages from human pluripotent stem cells under partly defined and fully defined conditions; 2013; PLOS One; Vol. 8, Issue 8.

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 phospho-SYK (Tyr525/526) 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. The following 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 phospho-SYK (Tyr525/526) detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF phospho-SYK (Tyr525/526) assay, 1,000 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-SYK assay was 4 times more sensitive than the Western Blot technique.