AlphaLISA SureFire Ultra Human Total CDK6 Detection Kit, 500 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.

Upregulation of CDK6 expression in MCF7 cells treated with NRG1

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 pretreated with 20 µM Wortmannin for 3 hours prior to treatment with increasing concentrations of Neuregulin1 (NRG1) for 24 hours. All treatments were performed in serum-free media.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking at 350 rpm. CDK4 and CDK6 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 induced a dose-dependent increase of CDK6.

CDK6 expression in various cell lines

Adherent cells were seeded at 50,000 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.

Suspension cells were seeded at 50,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA and lysed with 100 µL of Lysis Buffer. CDK6 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 5,000 cells) were 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.

CDK6 is expressed across a wide range of human cell types.