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

Phospho-AlphaLISA SureFire Biotin Free 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 Biotin Free 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 Biotin Free one-plate assay protocol

Detection of Phosphorylated target protein with AlphaLISA SureFire Biotin Free 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 robust AlphaLISA SureFire quality.

TREM2 Activator induces SYK phosphorylation in THP-1 derived macrophages

HP-1 cells were treated with 35 ng/mL TGF-ß for 18 hours at 37°C, 5% CO₂. Cells were seeded in a 96-well plate (400,000 cells/well) in serum free RPMI 1640 and treated with 20 µM TREM2 Activator (MedChemExpress, HY-148095) for 2 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). SYK Phospho (Tyr525/526) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. 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. The plate was read on an Envision using standard AlphaLISA settings.

TREM2 Activator triggered a rapid 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 (200,000 cells/well) in serum free RPMI 1640 and treated with increasing concentrations of Anti-IgM for 20 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). SYK Phospho (Tyr525/526) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. 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. The plate was read on an Envision using standard AlphaLISA settings.

Treatment with Anti-IgM 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 Pervanadate treated cells

U937 cells were seeded in a 96-well plate (400,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of Pervanadate for 30 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). SYK Phospho (Tyr525/526) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. 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. The plate was read on an Envision using standard AlphaLISA settings.

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