AlphaLISA SureFire Ultra Human and Mouse Phospho-STAT3 (Ser727) 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 enables cell 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 robust AlphaLISA SureFire Ultra quality.

Induction of STAT3 phosphorylation in EGF treated cells

HeLa cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were starved in media containing 1% FBS for 20 hours then treated with increasing concentrations of EGF for 15 minutes.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT3 Phospho (Ser727) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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, EGF triggered a dose-dependent increase in the levels of Phospho (Ser727) while Total STAT3 levels remained unchanged.

Induction of STAT3 phosphorylation in IL-6 treated cells

A431 cells were seeded in a 96-well plate (30,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were starved in medium containing 1% FBS for 20 hours then treated with increasing concentrations of IL-6 for 15 minutes.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT3 Phospho (Ser727) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 3,000 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, IL-6 treatment induced a dose-dependent increase in the levels of Phospho (Ser727) while Total STAT3 levels remained unchanged.

Induction of STAT3 phosphorylation in PMA treated cells

A431 cells were seeded in a 96-well plate (30,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were starved in media containing 1% FBS for 20 hours then treated with increasing concentrations of PMA for 30 minutes.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT3 Phospho (Ser727) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 3,000 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, PMA treatment induced a dose-dependent increase in the levels of Phospho (Ser727) while Total STAT3 levels remained unchanged.

Inhibition of STAT3 phosphorylation in Staurosporine treated cells

HeLa cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were starved in media containing 1% FBS for 20 hours then treated with 5 µM Staurosporine for 30 minutes.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT3 Phospho (Ser727) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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, Staurosporine decreased the levels of STAT3 Phospho (Ser727) while Total levels remained unchanged.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from A431 cells cultured to confluency in a T175 flasks, treated with 2 µg/mL EGF for 10 minutes and lysed in 5 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and STAT3 Phospho (Ser727) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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.

Approximate number of cells per datapoint is indicated. The dotted line represents assay background. The assay can detect STAT3 Phospho (Ser727) down to 50 cells/datapoint.