AlphaLISA SureFire Ultra Human and Mouse Phospho-MEK2 (Ser217/221) 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.

Activation of Phospho MEK2 (Ser217/221) in EGF treated cells

HEK293 cells were seeded in a 96-well plate (60,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were starved for 2 hours and then treated with increasing concentrations of EGF in serum free media for 10 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MEK2 Phospho (Ser217/221) and Total 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 MEK2 (Ser217/221) while Total MEK2 levels remained unchanged.

Activation of Phospho MEK2 (Ser217/221) in NRG1 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 2 hours and then treated with increasing concentrations of Neuregulin (NRG1) in serum free media for 10 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MEK2 Phospho (Ser217/221) 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, NRG1 triggered a dose-dependent increase in the levels of Phospho MEK2 (Ser217/221) while Total MEK2 levels remained unchanged.

Activation of Phospho MEK2 (Ser217/221) in anti-CD3 treated cells

Jurkat cells were washed and resuspended in HBSS + 0.1% BSA at a density of 2x10⁶ cells/mL, then seeded in a 96-well plate (200 µL per well). The cells were starved for 1 hour and then treated with increasing concentrations of anti-CD3 antibody in HBSS + 0.1% BSA for 5 minutes.

After treatment, the cells were lysed with 50 µL 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MEK2 Phospho (Ser217/221) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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 antibody triggered a dose-dependent increase in the levels of Phospho MEK2 (Ser217/221) while Total MEK2 levels were unchanged.

Decrease of Phospho MEK2 (Ser217/221) levels in AG1478 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 treated with increasing concentrations of AG1478 for 2 hours followed by treatment 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). MEK2 Phospho (Ser217/221) 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 (EGFR inhibitor) triggered a dose-dependent decrease in the levels of Phospho MEK2 (Ser217/221) while Total MEK2 levels remained unchanged.

Specificity of Phospho MEK2 assay – Recombinant protein

Specificity of the Phospho MEK2 (Ser217/221) assay was assessed by assaying recombinant MEK1 and MEK2 proteins. Dilutions of recombinant human MEK1 and MEK2 proteins were prepared in Lysis Buffer. Phospho MEK2 (Ser217/221) levels were evaluated using the AlphaLISA SureFire Ultra assay.

For the detection step, 10 µL of 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.

Phospho MEK2 (Ser217/221) assay was specific only to MEK2 recombinant protein with no signal detected with MEK1 recombinant protein. This result demonstrates the specificity of Phospho MEK2 (Ser217/221) AlphaLISA SureFire Ultra assay as these two proteins share more than 80% sequence identity.

Specificity of Phospho MEK2 assay in knockout cells

Phospho MEK2 (Ser217/222) protein levels were assessed in HEK293T wild type (WT) and HEK293T MEK2 knockout (KO) (Abcam ab266315) cells. Both cell lines, WT and KO, 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 2 hours and then treated with increasing concentrations of EGF in serum free media 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 MEK2 (Ser217/222) levels were then evaluated using the SureFire Ultra assay. For the detection step, 10 µL of cell lysate 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, Phospho MEK2 (Ser217/211) was only detected in the WT HEK293T cells but not in the MEK2 KO cell lines, demonstrating assay specificity.