Myeloid Cell Leukemia 1 (MCL-1) is an anti-apoptotic protein belonging to the BCL-2 family that prevents mitochondrial outer membrane permeabilization and cytochrome c release. MCL-1 localizes to the mitochondrial outer membrane where it sequesters pro-apoptotic proteins such as BAK, BAX, and BH3-only proteins, thereby blocking apoptosis initiation. MCL-1 expression is tightly regulated at transcriptional, post-transcriptional, and post-translational levels, with rapid turnover under normal conditions. Overexpression of MCL-1 is frequently observed in hematologic malignancies and solid tumors, where it confers resistance to chemotherapy and targeted therapies. MCL-1 dependency varies among cancer types, making it an attractive target for selective cancer therapy. BH3 mimetics and MCL-1-specific inhibitors are being developed to overcome apoptotic resistance in MCL-1-dependent cancers.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-MCL-1 (Thr163) is a sandwich immunoassay for the quantitative detection of phospho-MCL-1 (Thr163) in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
Phospho-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 Phospho-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Phospho-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Phosphorylated 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 robust 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.
Inhibition of MCL-1 phosphorylation in cells treated with HSP90 inhibitors
THP-1 cells were seeded in a 96-well plate (100,000 cells/well or 150,000 cells/well) in complete medium and treated with increasing concentrations of SNX-2112 or 17-AAG for 24 hours at 37°C, 5% CO2.
After treatment, the cells were washed with HBSS then 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 10,000 cells for SNX-2112 or 15,000 cells for 17-AAG treatment) 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.
HSP90 inhibitors, SNX-2112 and 17-AAG, triggered a dose-dependent decrease in the levels of MCL-1 Phospho (Thr163) while Total MCL-1 levels remained unchanged.
Knockout validation of MCL-1 Phospho (Thr163) assay
MCL-1 Phospho (Thr163) 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 Phospho (Thr163) 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 Phospho (Thr163) signal was only detected in WT cells confirming the specificity of the assay.
