AlphaLISA SureFire Ultra Human and Mouse Phospho-ERK1/2 (Thr202/Tyr204) 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 ERK1/2 (Thr202/Tyr204) phosphorylation in primary cells

PBMCs were isolated from healthy donors using Ficoll Plaque Plus (Merck, GE17-1440-02), Cells were seeded in a 96-well plate (400,000 cells/well) in complete DMEM and treated with the indicated agonists for 30 minutes.

After treatment, the cells were lysed with 400 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ERK1/2 Phospho (Thr202/Tyr204) levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 10,000 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, ERK phosphorylation was induced by PMA and LPS in PBMCs.

Activation of Phospho ERK1/2 (Thr202/Tyr204) in Anti-CD3 treated cells

Jurkat cells were seeded in a 96-well plate (50,000 cells/well) in HBSS + 0.1% BSA. The cells were starved for 60 minutes and then treated with increasing concentrations of Anti-CD3 antibody for 5 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). ERK1/2 Phospho (Thr202/Tyr204) 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, Anti-CD3 triggered a dose-dependent increase in the levels of Phospho ERK1/2 (Thr202/Tyr204) while Total ERK1/2 levels remained unchanged.

Activation of Phospho ERK1/2 (Thr202/Tyr204) in Neuregulin1 treated cells

MCF7 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 starved for two hours and then treated with increasing concentrations of Neuregulin1 (NRG1) for 10 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ERK1/2 Phospho (Thr202/Tyr204) and Total 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, Neuregulin1 triggered a dose-dependent increase in the levels of Phospho ERK1/2 (Thr202/Tyr204) while Total ERK1/2 levels remained unchanged.

Activation of Phospho ERK1/2 (Thr202/Tyr204) in EGF treated cells

HEK293 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 starved for two hours and then treated with increasing concentrations of EGF for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ERK1/2 Phospho (Thr202/Tyr204) and Total 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 triggered a dose-dependent increase in the levels of Phospho ERK1/2 (Thr202/Tyr204) while Total ERK1/2 levels remained unchanged.

Activation of Phospho ERK1/2 (Thr202/Tyr204) in TNFα treated cells

HeLa 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 TNFα for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ERK1/2 Phospho (Thr202/Tyr204) and Total 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, TNFα triggered a dose-dependent increase in the levels of Phospho ERK1/2 (Thr202/Tyr204) while Total ERK1/2 levels remained unchanged.

Decrease of Phospho ERK1/2 (Thr202/Tyr204) levels in AG1478 treated cells

HeLa 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 AG1478 for 2 hours, and then 1 ng/mL EGF for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ERK1/2 Phospho (Thr202/Tyr204) and Total 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, AG1478 triggered a dose-dependent decrease in the levels of Phospho ERK (Thr202/Tyr204) while Total ERK1/2 levels remained unchanged.

Decrease of Phospho ERK1/2 (Thr202/Tyr204) levels in Oxozeanol treated cells

HEK293 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 Oxozeanol for 2 hours followed by treatment with 0.75M Sorbitol for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ERK1/2 Phospho (Thr202/Tyr204) and Total 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, Oxozeanol triggered a dose-dependent decrease in the levels of Phospho ERK (Thr202/Tyr204) while Total ERK1/2 levels remained unchanged.

Decrease of Phospho ERK1/2 (Thr202/Tyr204) levels in AMG-510 treated cells

SW 1573 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 AMG-510 for 1 hour.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). ERK1/2 Phospho (Thr202/Tyr204) and Total 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, AMG-510 triggered a dose-dependent decrease in the levels of Phospho ERK (Thr202/Tyr204) while Total ERK1/2 levels remained unchanged.