AlphaLISA SureFire Ultra Human and Mouse Phospho-MEK1 (Ser218/222) 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.

Activation of Phospho MEK1 (Ser218/222) 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 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). Phospho (Ser218/222) and Total MEK1 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, NRG1 triggered a dose-dependent increase in the levels of Phospho (Ser218/222) MEK1 while Total levels remained unchanged.

Activation of Phospho MEK1 (Ser218/222) in EGF treated cells

HEK293 cells were seeded in a 96-well plate (30,000 cells/well) in complete medium, and incubated for 48 hours at 37°C, 5% CO2. The cells were starved for 2 hours and treated 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 (Ser218/222) and Total MEK1 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, EGF triggered a dose-dependent increase in the levels of Phospho (Ser218/222) MEK1 while Total levels remained unchanged.

Activation of Phospho MEK1 (Ser218/222) 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). Phospho (Ser218/222) and Total MEK1 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 (Ser218/222) MEK1 while Total levels remained unchanged.

Decrease of Phospho MEK1 (Ser218/222) levels in AG1478 treated cells

A431 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated for 48 hours at 37°C, 5% CO2. The cells were treated with increasing concentrations of AG1478 for 2 hours and then treated with 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). Phospho (Ser218/222) and Total MEK1 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 (Ser218/222) MEK1 stimulated by EGF, while Total levels remained unchanged.

Decrease of Phospho MEK1 (Ser218/222) 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 2 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Ser218/222) and Total MEK1 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 a KRAS G12C inhibitor, triggered a dose-dependent decrease in the levels of Phospho (Ser218/222) MEK1 while Total levels remained unchanged.

MEK1 Phospho (Ser218/222) Assay specificity

Specificity of the MEK1 Phospho (Ser218/222) assay was assessed by using active MEK1 and MEK2 recombinant proteins.

Dilutions of recombinant MEK1 (Abcam, ab63209) and MEK2 (Merck, SLBN1818V) proteins were prepared in Lysis Buffer and evaluated using the AlphaLISA SureFire Ultra assay.

For the detection step, 10 µL of diluted protein 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.

The MEK1 Phospho (Ser218/222) assay showed reactivity only to MEK1 protein, no cross reactivity to MEK2 protein was observed.