AlphaLISA SureFire Ultra Human & Mouse Total CREBBP 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.

Degradation of CREBBP in PROTAC treated cells

HeLa 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 d-CBP for 2 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking at 350 rpm. CREBBP 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, d-CBP induced a dose-dependent decrease in CREBBP levels.

Specificity of CREBBP Total assay

Total CREBBP protein levels were assessed in HEK293T cells (WT) and CREBBP knockout (KO) HEK293T cells. CREBBP KO cells (Abcam ab266099) and WT cells were seeded in a 96-well plate in complete medium, and incubated overnight at 37°C, 5% CO2.

Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CREBBP Total levels were then evaluated by AlphaLISA SureFire Ultra. 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.

CREBBP was detected in the WT cells but not in the CREBBP-KO cell line, demonstrating that the Total CREBBP kit specifically measures CREBBP and does not recognize other family members.

Versatility of Total CREBBP assay in various cell lines

Adherent cells were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2. Cells were lysed with 50 µL of Lysis Buffer. 
Suspension cells were seeded at 40,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA and lysed with 50 µL of Lysis Buffer.

CREBBP Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 8,000 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.

CREBBP is expressed across a wide range of human and mouse cell types.