AlphaLISA SureFire Ultra Human Total p21 CiP1 Detection Kit, 10,000 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.

Activation of total p21 CiP1 in Doxorubicin treated cells

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 treated with increasing concentrations of Doxorubicin for 18 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). p21 CiP1 Total and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 cells and approximately 80 cells for Cofilin Total) 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, Doxorubicin triggered a dose-dependent increase in the levels of Total p21 CiP1 while Total Cofilin levels remained unchanged.

Decrease of total p21 CiP1 in Thapsigargin treated cells

SH-SY5Y 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 Thapsigargin for 3 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). p21 CiP1 Total and GAPDH 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.

Thapsigargin triggered a dose-dependent decrease in the levels of p21 CiP1 Total in SH-SY5Y cells while Total GAPDH levels remained unchanged.

Differential expression of p21 CiP1 total in human and mouse cell lines

Human and mouse cells lines were seeded at 40,000 cells/well in a 96-well culture plate in complete medium and incubated overnight at 37°C, 5% CO2. The cells were lysed the following day with 100 µL of Lysis Buffer.

p21 CiP1 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL (approximately 4,000 cells) of cell lysate 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.

Total p21 CiP1 expression was detected across all human and mouse adherent cell lines tested. A very high level of expression was detected in HeLa, HEK293 and SW 1573 cells, with lower expression in A431, SH-SY5Y, HEK293T, RAW 264.7 and NIH/3T3 cells, demonstrating Total p21 CiP1 assay versatility.