AlphaLISA SureFire Biotin-Free High Performance Human Phospho-STAT6 (Tyr641) Detection Kit, 500 assay points

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

Detection of Phosphorylated target protein with AlphaLISA SureFire Ultra 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 Ultra quality.

STAT6 phosphorylation in THP-1 derived macrophages

THP-1 cells were seeded in a 96-well plate (100,000 cells/well) in complete RPMI 1640 medium containing 100 nM of PMA for 24 hours at 37°C, 5% CO2. The THP-1 derived macrophages were starved for 2 hours and then treated with various cytokines for 15 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT6 Phospho (Tyr641) and Total levels were evaluated using AlphaLISA SureFire Biotin Free Phospho (Tyr641) (HP) and Total STAT6 assays. For the detection step, 10 µL of cell lysate (approximately 10,000 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, IL-4 induced the greatest increase in the levels of STAT6 Phospho (Tyr641).

Activation of STAT6 Phospho (Tyr641) in THP-1 cells

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete RPMI 1640 medium and treated with increasing concentrations of IL-4 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). STAT6 Phospho (Tyr641) and Total levels were evaluated using SureFire Biotin Free kits ASBF-PST6-A and ASBF-PST6-B, and ASBF-TST6-A.

For the detection step, lysate was further diluted 1:4 with Lysis Buffer, 10 µL of cell lysate (approximately 2,000 cells/datapoint) 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, IL-4 triggered a dose-dependent increase in the levels of Phospho (Tyr641) STAT6 while Total levels remained unchanged.