AlphaLISA SureFire Ultra Human Total Cyclin E1 Detection Kit, 100 Assay Points

Total-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 Total-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.

Total-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Total 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.

Hydroxyurea induction of Cyclin E1

A549 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated for 24 hours at 37°C, 5% CO2. 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). Phospho (Thr77) and Total Cyclin E1 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, Hydroxyurea treatment resulted in a dose-dependent increase in the levels of Phospho (Thr77) and Total Cyclin E1.

Upregulation of Cyclin E1 by Neuregulin-1

MCF7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium, and incubated for 24 hours at 37°C, 5% CO2. Cells were treated with 20 µM wortmannin for 3 hours and then increasing concentrations of Neuregulin-1 for 24 hours in serum free medium.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Total Cyclin E1, Phospho (Thr160) and Total CDK2 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, Neuregulin-1 treatment resulted in a dose-dependent increase in Cyclin E1 levels as well as activation of other cell cycle regulators.

A549 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium, and incubated for 24 hours at 37°C, 5% CO2. 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). Phospho (Thr77) and Total Cyclin E1 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, Nocodazole treatment induced a dose dependent decrease in the levels of Phospho (Thr77) and Total Cyclin E1.

A431 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium and incubated for 24 hours at 37°C, 5% CO2. 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). Levels of Cyclin E1 and Cofilin were evaluated using respective AlphaLISA SureFire Ultra assays. 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, Nocodazole treatment induced a dose dependent decrease in the levels of Cyclin E1 with no changes in the levels of Cofilin.

Versatility of Total Cyclin E1 assay in various cell lines

Adherent cells were seeded at 20,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2. Cells were lysed with 100 µL of Lysis Buffer.

Cyclin E1 levels were evaluated by AlphaLISA SureFire Ultra. 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, Cyclin E1 is expressed at high levels in BeWo cells.