AlphaLISA SureFire Ultra Human Phospho-EGFR (Tyr1068) Detection Kit, 10,000 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 phosphorylation in A431 EGF treated cells

A431 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 serum starved for 2 hours and then treated in serum free medium with increasing concentrations of EGF for 10 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr1068) and Total EGFR 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, EGF induced a dose-dependent increase in Phospho (Tyr1068) EGFR levels while Total levels remained unchanged.

AG1478 inhibits EGFR phosphorylation in A431 cells

A431 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 treated with increasing concentrations of AG1478 for 2 hours in serum free media. After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr1068) and Total EGFR 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, AG1478 decreased Phospho (Tyr1068) EGFR levels in a dose-dependent manner while Total levels remained unchanged.

Rociletinib inhibits EGFR phosphorylation in NCI-H1975 cells

NCI-H1975 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 increasing concentrations of Rociletinib for 24 hours. After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr1068) and Total EGFR 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, Rociletinib decreased Phospho (Tyr1068) EGFR levels in a dose-dependent manner while Total levels remained unchanged.

EGFR assay sensitivity - cell lysate dilution

Cell lysate was prepared from A431 cells cultured to confluence in T175 flasks in complete medium. Cells were treated with 2 µg/mL EGF for 10 minutes and then lysed in 8 mL of Lysis Buffer.

Lysates were serially diluted in Lysis Buffer and assayed for Phospho (Tyr1068) and Total EGFR levels using respective AlphaLISA SureFire Ultra kits. 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/datapoint is indicated. The dotted line represents assay background. The assay can detect EGFR protein down to 50 cells/datapoint.