Description
Signal Transducer and Activator of Transcription 4 (STAT4) is a transcription factor that mediates cellular responses to IL-12 and type I interferons, playing essential roles in Th1 cell differentiation and NK cell activation. STAT4 is activated by phosphorylation at Tyr693, leading to dimerization and nuclear translocation. Activated STAT4 induces expression of IFN-γ and T-bet, promoting Th1 responses and cell-mediated immunity against intracellular pathogens. STAT4 contributes to autoimmune disease pathogenesis, with genetic variants associated with increased risk of rheumatoid arthritis and lupus. Modulating STAT4 activity represents a therapeutic opportunity for enhancing immunity or dampening pathological Th1 responses.
The AlphaLISA SureFire Biotin-Free Human Phospho-STAT4 (Tyr693) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-STAT4 (Tyr693) 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 Biotin Free kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Biotin rich samples
AlphaLISA SureFire Biotin Free kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho-AlphaLISA SureFire Biotin Free assay principle
The Phospho-AlphaLISA SureFire Biotin Free assay measures a protein target 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 a proprietary CaptSure3 agent to capture one of the detection antibodies, which is labeled with CaptSure3 tag. 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 Biotin Free 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 Biotin Free 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 Biotin Free one-plate assay protocol
Detection of Phosphorylated target protein with AlphaLISA SureFire Biotin Free 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 quality.
Activation of phospho STAT4 (Tyr693) in NK-92 cells
NK-92 cells were seeded in a T175 flask at 500K/mL in complete MEMα medium deprived of IL-2 and incubated overnight. Cells were seeded in a 96-well plate (500,000 cells/well) and treated with increasing concentrations of IL-12 in serum free RPMI 1640 for 90 minutes.
After treatment, the cells were lysed with the addition of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT4 Phospho (Tyr693) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free 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, IL-12 triggered a dose-dependent increase in the levels of STAT4 Phospho (Tyr693) while Total levels remained unchanged.
Assay sensitivity - cell lysate
Cell lysate was prepared from NK-92 cells IL-2 starved for 18 hours and stimulated with 200 ng/mL IL-12 for 30 minutes in HBSS + 0.1% BSA. Cells were lysed at 3.2 x 106 with the addition of 5X Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and STAT4 Phospho (Tyr693) and Total levels were evaluated with AlphaLISA SureFire Biotin Free assays. 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. This assay can detect STAT4 Phospho (Tyr693) down to 500 cells/datapoint.
