Revvity, ALSU-TFOXO3-A500, AlphaLISA Human and Mouse Total FOXO3A Detection Kit, 500 Assay Points

Overview
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 Total FOXO3A is a sandwich immunoassay for the quantitative detection of total FOXO3A 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
How it works
Total-AlphaLISA SureFire Ultra assay principle
The Total-AlphaLISA SureFire Ultra assay measures the expression level of a protein target in a cell lysate.
The Total-AlphaLISA SureFire Ultra assay uses two antibodies which recognize two different distal epitopes on the targeted 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 targeted 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 the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of protein present in the sample.

 

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.
Assay versatility
Expression of FOXO3A in various cell lines
Adherent cell lines were seeded in a 96-well plate (40,000 cells/well) and incubated overnight at 37°C, 5% CO2. Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).
Suspension cell lines were seeded in a 96-well plate (400,000 cells/well) and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).
FOXO3A levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells and 40,000 suspension cells) were 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.
Total FOXO3A protein is expressed in a wide range of cell types, with particularly high expression in HEK293, RPMI 8226 and mouse cell line NIH/3T3.
Application-Cell Signaling
Automation Compatible-Yes
Brand-AlphaLISA SureFire Ultra
Detection Modality-Alpha
Protocol Time-2h at RT
Sample Volume-10 µL
Shipping Conditions-Shipped in Blue Ice
Target-FOXO3A
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-Alpha
Therapeutic Area-Neuroscience Oncology
Unit Size-500 assay points