AlphaLISA SureFire Ultra Human and Mouse Total MCL-1 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.

Induction of MCL-1 phosphorylation in EGF treated cells

HeLa cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of EGF for 2 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MCL-1 Phospho (Thr163) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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, the EGF triggered a significant increase in the levels of Phospho (Thr163) and a modest increase in Total MCL-1 levels.

Induction of MCL-1 phosphorylation in PMA treated cells

HeLa cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of PMA for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MCL-1 Phospho (Thr163) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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, the PMA triggered a dose-dependent increase in the levels of Phospho (Thr163) MCL-1 while MCL-1 Total remained unchanged.

Induction of MCL-1 phosphorylation in PMA treated cells

Raji cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and treated with increasing concentrations of PMA for 30 minutes at 37°C, 5% CO₂.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MCL-1 Phospho (Thr163) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 20,000 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, the PMA triggered a dose-dependent increase in the levels of Phospho (Thr163) MCL-1 while MCL-1 Total remained unchanged.

Knockout validation of MCL-1 Total assay

MCL-1 Total levels were assessed in HEK293T Wild Type (WT) and MCL-1 knockout (KO) cells (Abcam ab266838). Cells were seeded at various densities in a 96 well plate in complete medium, and incubated overnight at 37°C, 5% CO₂.

The cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). MCL-1 levels were 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.

MCL-1 signal was only detected in the WT cells confirming the specificity of the assay.

Expression of MCL-1 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 200 µL of Lysis Buffer.

Suspension cells were seeded at 400,000 cells/well in a 96-well culture plate in HBSS + 0.1% BSA, cells were spun down and lysed with 200 µL of Lysis Buffer. MCL-1 Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 2,000 adherent cells or 20,000 suspension 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.

MCL-1 is broadly expressed in various cell lines. High MCL-1 expression in observed in Raji and EL-4 cells while lower levels were detected in Hep G2 and K-562 cells.