AlphaLISA SureFire Ultra Human and Mouse Phospho-4EBP1 (Ser65) 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.

Inhibition of 4E-BP1 phosphorylation in LY294002 treated cells

MCF7 cells were seeded in a 96-well plate (10,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of LY294002 for 2 hours in serum-free media.

After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). 4E-BP1 Phospho (Ser65) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. Cell lysate was further diluted in Lysis Buffer. For the detection step, 10 µL of cell lysate (approximately 250 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, treatment with LY294002 resulted in a dose-dependent decrease of Phospho (Ser65) 4E-BP1 levels and a modest decrease in Total levels, while Total ERK levels were unchanged (data not shown).

Inhibition of 4E-BP1 phosphorylation in Torin treated cells

MCF7 were seeded in a 96-well plate (10,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of Torin for 1 hour.

After treatment, cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). 4E-BP1 Phospho (Ser65) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. Cell lysate was further diluted in Lysis Buffer. For the detection step, 10 µL of cell lysate (approximately 250 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, treatment with Torin resulted in a dose-dependent decrease of Phospho (Ser65) 4E-BP1 levels while Total levels remained unchanged.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from MCF7 cells cultured to confluency in a T175 flasks, treated with 500 µg/mL Insulin for 30 minutes and lysed in 20 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and Phospho (Ser65) 4E-BP1 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 Phospho (Ser65) 4E-BP1 down to 30 cells/datapoint.