AlphaLISA SureFire Ultra Human and Mouse Phospho-TYK2 (Tyr1054/1055) Detection Kit, 100 Assay Points

Phospho-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.

Induction of TYK2 (Tyr1054/1055) phosphorylation in primary cells

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 starved for 2 hours and then treated with the indicated concentrations of IFNα for 20 minutes.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr1045/1055) and Total TYK2 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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, IFNα triggered a dose-dependent increase in the levels of Phospho (Tyr1054/1055) TYK2 while Total levels remained unchanged.

PC3 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated for 24 hours at 37°C, 5% CO2. The cells were treated with increasing concentrations of IFNα for 15 minutes.

After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr1054/1055) and Total TYK2 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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, IFNα triggered a dose-dependent increase in the levels of Phospho (Tyr1054/1055) TYK2 while Total levels remained unchanged.

HEL 92.1.7 cells were seeded in a 96-well plate (500,000 cells/well) in HBSS + 0.1% BSA and treated with increasing concentrations of IFNα 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 (Tyr1054/1055) and Total TYK2 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 20,000 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, IFNα triggered a dose-dependent increase in the levels of Phospho (Tyr1054/1055) TYK2 while Total levels remained unchanged.

Specificity of Phospho TYK2 (Tyr1054/1055) assay

TYK2 (Tyr1054/1055) levels were assessed in Wild Type (WT) and TYK2 knockout (KO) HEK293T cells. TYK2 KO cells (Abcam ab266730) and WT HEK293T cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with 50 ng/mL IFNα for 10 minutes.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho TYK2 (Tyr1054/1055) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 2,000 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, TYK2 (Tyr1054/1055) was detected in the WT HEK293T cells treated with IFNα but not in the TYK2 KO cell line.