AlphaLISA SureFire Biotin-Free Human Total Cyclin E1 Detection Kit, 10,000 Assay Points

OVCAR3 cells were seeded in a 96-well plate (40,000 cells/well) in medium containing 20% FBS and 10 µg/mL insulin and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of hydroxyurea for 18 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Cyclin E1 Phospho (Thr77) and Cyclin E1 Total levels were evaluated using respective AlphaLISA SureFire Biotin Free 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, the hydroxyurea triggered a dose-dependent increase in the levels of Phospho (Thr77) and Total Cyclin E1.

A549 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 hydroxyurea for 18 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Cyclin E1 Phospho (Thr77) and Cyclin E1 Total levels were evaluated using respective AlphaLISA SureFire Biotin Free 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, hydroxyurea triggered a dose-dependent increase in the levels of Phospho (Thr77) and  Total Cyclin E1.

Inhibition of Cyclin E1 in nocodazole treated cells

A549 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 nocodazole for 18 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Cyclin E1 Phospho (Thr77) and Cyclin E1 Total levels were evaluated using respective AlphaLISA SureFire Biotin Free 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, treatment with nocodazole resulted in a dose-dependent decrease in the levels of Phospho (Thr77) and Total Cyclin E1.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from BeWo cells cultured to confluency in T175 flasks at 37°C, 5% CO₂. Each flask was lysed in 3 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and Cyclin E1 Phospho (Thr77) and Total levels were evaluated by AlphaLISA SureFire Biotin Free. 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. This assay can detect Cyclin E1 Total down to 100 cells/datapoint.