AlphaLISA SureFire Ultra Human and Mouse Phospho-SYK (Tyr525/526) Detection Kit, 500 Assay Points

AlphaLISA SureFire Ultra two-plate assay protocol

The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Phospho-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells' viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

Phospho-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Phosphorylated target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining AlphaLISA SureFire Ultra quality.

TREM2 Activator rapidly induces SYK phosphorylation in THP-1 and primary macrophages

THP-1 cells were treated with 35 ng/mL TGFb  for 18 hours at 37°C, 5% CO2. Cells were harvested and seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA and then treated with 20 µM TREM2 Activator (MedChemExpress, Cat # HY-148095) for various time points.

After treatment, cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). SYK Phospho (Tyr525/526) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 16,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark.

TREM2 activator induced SYK phosphorylation within just 2 minutes, peaking at 10 minutes of treatment.

PBMCs were isolated from healthy donors and cultured for 6 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (40,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO2. Cells were treated with 35 ng/mL TGFb for 18 hours and then treated with 20 µM TREM2 Activator (MedChemExpress, Cat # HY-148095) for various time points.

After treatment, cells were lysed in 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). SYK Phospho (Tyr525/526) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 8,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark.

As expected, TREM2 activator induced SYK phosphorylation very quickly, peaking at 10 minutes post- treatment.

Concanavalin A induces SYK phosphorylation in THP-1 cells

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in HBSS + 0.1% BSA and treated with increasing concentrations of Concanavalin A for 10 minutes.

After treatment, the cells were spun down at 300 RCF for 5 minutes, treatment removed and then lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr525/526) and Total SYK levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 20,000) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, Concanavalin A triggered a dose-dependent increase in the levels of Phospho SYK (Tyr525/526) while Total SYK levels remained unchanged.

Activation of Phospho SYK (Tyr525/526) levels in Anti-IgM treated cells

Ramos cells were seeded in a 96-well plate (300,000 cells/well) in HBSS + 0.1% HBSS and treated with 20 µg/mL Anti-IgM for 15 minutes.

After treatment, the cells were lysed with the addition of 50 µL of 5x Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr525/526) SYK levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 12,000 cells was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, Anti-IgM triggered an increase in the levels of Phospho (Tyr525/526) SYK.