Forkhead Box O3A (FOXO3A) is a transcription factor that regulates genes involved in apoptosis, cell cycle arrest, DNA repair, and stress resistance. FOXO3A activity is primarily controlled by AKT-mediated phosphorylation, which promotes its cytoplasmic sequestration and inactivation. Under stress conditions or reduced AKT signaling, FOXO3A translocates to the nucleus and activates target genes including p21, p27Kip1, Bim, and GADD45. FOXO3A functions as a tumor suppressor by promoting apoptosis and cell cycle checkpoints in response to oncogenic stress. Loss of FOXO3A activity through AKT hyperactivation contributes to cancer progression, chemoresistance, and metabolic reprogramming. Conversely, FOXO3A activation is associated with longevity and stress tolerance. Therapeutic strategies aim to restore FOXO3A function through AKT inhibition or direct FOXO3A activation in cancer treatment.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-FOXO3A (Ser253) is a sandwich immunoassay for the quantitative detection of phospho-FOXO3A (Ser253) 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.
Insulin induces FOXO3A phosphorylation in a dose-dependent manner
MCF7 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 starved for 2 hours and then treated with increasing concentrations of insulin for 5 minutes prepared in serum free medium.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). FOXO3A Phospho (Ser253) 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, insulin triggered a dose-dependent increase in the levels of Phospho FOXO3A (Ser253) with a modest increase in Total levels (2.7-fold).
NIH/3T3 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 starved for 2 hours and then treated with increasing concentrations of insulin prepared in serum free medium for 30 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). FOXO3A Phospho (Ser253) and 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, insulin triggered a dose-dependent increase in the levels of Phospho FOXO3A (Ser253) while total levels did not increase significantly.
Insulin growth factor induces FOXO3A phosphorylation in MCF7 cells
MCF7 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 starved for 2 hours and treated with 500 ng/mL IGF-1 for 5 minutes in serum free medium.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). FOXO3A Phospho (Ser253) levels were evaluated using the AlphaLISA SureFire Ultra assay. 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, treatment with IGF-1 triggered a significant increase in the levels of phosphorylated FOXO3A (Ser253) in MCF7 cells with no change to Total FOXO3A levels (not shown).
PI3K inhibition of FOXO3A phosphorylation
HEK293 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 20 µM of LY294002 for 24 hours.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). FOXO3A Phospho (Ser253) levels were evaluated using the AlphaLISA SureFire Ultra assay. 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, LY294002 triggered a significant decrease of phosphorylated FOXO3A at Ser253.
Knockout validation of Phospho FOXO3A (Ser253) assay
Phospho FOXO3A (Ser253) levels were assessed in HEK293 wild type (WT) and HEK293 FOXO3A knockout (KO) cells (Abcam, ab260857). Cells were cultured to confluency in T75 flasks at 37°C, 5% CO2. Cells were treated with 80 nM Calyculin A for 1 hour and then lysed in 2 mL of Lysis Buffer.
Lysates were serially diluted in Lysis Buffer and FOXO3A Phospho (Ser253) levels were evaluated using the AlphaLISA SureFire Ultra kit.
For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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.
Phospho FOXO3A (Ser253) was only detected in WT cells, confirming assay selectivity.
