AlphaLISA SureFire Ultra Human Phospho-JAK1 (Tyr1034/1035) Detection Kit, 500 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 JAK1 (Tyr1034/1035) phosphorylation in primary macrophages

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 (30,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of IFNα for 10 minutes.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JAK1 Phospho (Tyr1034/1035) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 6,000 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 JAK1 (Tyr1034/1035) in primary macrophages.

THP-1 cells were seeded in a 96-well plate (100,000 cells/well) in RPMI 1640 complete medium containing 100 nM PMA and incubated for 24 hours at 37°C, 5% CO₂. The THP-1 derived macrophages were washed and treated with increasing concentrations of IFNα for 10 minutes in HBSS + 0.1% BSA.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JAK1 Phospho (Tyr1034/1035) and Total 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 JAK1 (Tyr1034/1035) while Total JAK1 levels remained unchanged.

HEL92.1.7 cells were washed and seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA and treated with increasing concentrations of IFNα for 10 minutes.

After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JAK1 Phospho (Tyr1034/1035) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 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.

HEL92.1.7 cells were washed and seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA. The cells were stimulated with 100 ng/mL IFNα for 10 minutes and then treated with increasing concentrations of Ruxolitinib for 15 minutes.

After treatment, the cells were spun down and lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JAK1 Phospho (Tyr1034/1035) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 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.

Ruxolitinib (JAK1/2 inhibitor) induced a dose-dependent decrease in the levels of Phospho JAK1 (Tyr1034/1035), while JAK1 total levels remained unchanged.

THP-1 cells were seeded in a 96-well plate (100,000 cells/well) in complete medium containing 100 nM PMA and incubated for 24 hours at 37°C, 5% CO₂. The THP-1 derived macrophages were washed and stimulated with 100 ng/mL IFNα for 10 minutes followed by treatment with increasing concentrations of Ruxolitinib for 15 minutes. All treatments were performed in HBSS + 0.1% BSA.

After treatment, the cells were lysed with 50 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JAK1 Phospho (Tyr1034/1035) and Total 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.