Forkhead Box O1 (FOXO1) is a transcription factor that regulates genes involved in glucose metabolism, cell cycle arrest, apoptosis, and oxidative stress resistance. FOXO1 activity is controlled by AKT-mediated phosphorylation, which promotes nuclear exclusion and proteasomal degradation. Dephosphorylated FOXO1 translocates to the nucleus and activates target genes including glucose-6-phosphatase, PEPCK, and p27Kip1. FOXO1 plays critical roles in hepatic gluconeogenesis and functions as a tumor suppressor in cancer. Dysregulation of FOXO1 is implicated in type 2 diabetes and cancer, making it a therapeutic target for metabolic disorders and malignancies.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-FOXO1 (Ser256) is a sandwich immunoassay for the quantitative detection of phospho-FOXO1 (Ser256) 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 FOXO1 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 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). FOXO1 Phospho (Ser256) 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 FOXO1 (Ser256) with no significant changes in Total levels.
Razuprotafib induces FOXO1 phosphorylation in HUVEC cells
HUVEC 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 increasing concentrations of Razuprotafib for 15 minutes.
After treatment, the cells were lysed with 25 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). FOXO1 Phospho (Ser256) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 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.
Razuprotafib treatment results in the activation of the Tie2 signaling pathway in endothelial cells and a dose-dependent increase in the levels of Phospho FOXO1 (Ser256) with no significant changes in Total levels.
Insulin growth factor induces FOXO1 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 500 ng/mL of IGF-1 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). FOXO1 Phospho (Ser256) 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, IGF-1 increased the levels of Phospho FOXO1 (Ser256) with no significant changes in Total levels (data not shown).
Decrease in FOXO1 phosphorylation by PI3K inhibitor
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 50 µM 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). FOXO1 Phospho (Ser256) levels were evaluated using the AlphaLISA SureFire Ultra assay. 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, LY294002 treatment resulted in a decrease of phosphorylated FOXO1 at Ser256.
Knockout validation of Phospho FOXO1 (Ser256) assay
Phospho FOXO1 (Ser256) levels were assessed in HAP1 wild type (WT) and FOXO1 knockout (KO) cells. Cells were cultured to confluency in T175 flasks at 37°C, 5% CO2. Cells were lysed in 4 mL of Lysis Buffer.
Lysates were serially diluted in Lysis Buffer and FOXO1 Phospho (Ser256) 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 FOXO1 (Ser256) was only detected in WT cells, confirming assay specificity.
Assay sensitivity - cell lysate
Cell lysate was prepared from HEK293 cells cultured to confluency in a T175 flask and lysed in 3 mL of Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and assayed for Phospho (Ser256) and Total FOXO1 levels using respective AlphaLISA SureFire Ultra kits. 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.
Approximate number of cells/datapoint is indicated on the graph. The dotted line represents assay background. This assay can detect Phospho (Ser256) in approximately 2,500 cells/datapoint.
