Description
Eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) is a translational repressor that regulates cap-dependent protein synthesis by binding and sequestering eIF4E, preventing formation of the eIF4F translation initiation complex. 4EBP1 activity is controlled by mTORC1-mediated phosphorylation, with hyperphosphorylation causing release of eIF4E and activation of translation. Hypophosphorylated 4EBP1 preferentially inhibits translation of mRNAs encoding oncogenic proteins including c-MYC, cyclin D1, and VEGF. Dysregulation of 4EBP1 through mTOR pathway hyperactivation is common in cancer, promoting tumor progression and metabolic disorders. Therapeutic strategies targeting the mTOR-4EBP1 axis aim to restore translational control in cancer and metabolic diseases.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-4EBP1 (Ser65) is a sandwich immunoassay for the quantitative detection of phospho-4EBP1 (Ser65) 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.
Inhibition of 4E-BP1 phosphorylation in LY294002 treated cells
MCF7 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 increasing concentrations of LY294002 for 2 hours in serum-free media.
After treatment, the cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). 4E-BP1 Phospho (Ser65) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. Cell lysate was further diluted in Lysis Buffer. For the detection step, 10 µL of cell lysate (approximately 250 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 LY294002 resulted in a dose-dependent decrease of Phospho (Ser65) 4E-BP1 levels and a modest decrease in Total levels, while Total ERK levels were unchanged (data not shown).
Inhibition of 4E-BP1 phosphorylation in Torin treated cells
MCF7 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 increasing concentrations of Torin for 1 hour.
After treatment, cells were lysed with 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). 4E-BP1 Phospho (Ser65) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. Cell lysate was further diluted in Lysis Buffer. For the detection step, 10 µL of cell lysate (approximately 250 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 Torin resulted in a dose-dependent decrease of Phospho (Ser65) 4E-BP1 levels while Total levels remained unchanged.
Assay sensitivity - cell lysate dilution
Cell lysate was prepared from MCF7 cells cultured to confluency in a T175 flasks, treated with 500 µg/mL Insulin for 30 minutes and lysed in 20 mL of Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and Phospho (Ser65) 4E-BP1 levels were evaluated by AlphaLISA SureFire Ultra. 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 per datapoint is indicated. The dotted line represents assay background. The assay can detect Phospho (Ser65) 4E-BP1 down to 30 cells/datapoint.
