AlphaLISA SureFire Ultra Human Total p16 INK4A 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.

Increase of total p16 INK4A levels in Palbociclib treated cells

HEK293 cells were seeded in a 96-well plate (10,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with 10 µM of Palbociclib for 24 hours. After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Lysates were further diluted 1:50 in Lysis Buffer before proceeding to the detection step. Total p16 INK4A levels were evaluated using AlphaLISA SureFire Ultra assay kit.

For the detection step, 10 µL of cell lysate (approximately 20 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.

Palbociclib triggered an increase in Total p16 INK4A levels in HEK293 cells. Total Cofilin levels remain unchanged (data not shown).

Specificity of total p16 INK4A assay

Specificity of the Total p16 INK4A assay was assessed by assaying recombinant p16 INK4A and p15 INK4b protein. Dilutions of recombinant human p16 INK4A (Abcam, ab84075) and p15 INK4B (Abcam, ab268830) proteins were prepared in Lysis Buffer. p16 INK4A Total levels were evaluated using the AlphaLISA SureFire Ultra assay.

For the detection step, 10 µL of protein 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.

Total p16 INK4A assay was specific only to p16 INK4A recombinant protein with no signal detected with p15 INK4B recombinant protein. This result demonstrates the specificity of the Total p16 INK4A AlphaLISA SureFire Ultra assay as these two proteins share more than 80 % sequence identity.

Differential expression of p16 INK4A total in various human cell lines

Human cell lysates were diluted with Lysis Buffer. Approximate number of cells/datapoint is indicated on graph. Total p16 INK4A levels were evaluated using the AlphaLISA SureFire Ultra assay kit. 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.

Total p16 INK4A expression is dependent upon cell type. As expected, a very high level of expression was detected in HEK293 and HeLa cells, with lower expression in Hep G2, SH-SY5Y and BeWo cells.