Signal Transducer and Activator of Transcription 2 (STAT2) is a transcription factor that primarily mediates type I and type III interferon (IFN) signaling, playing a central role in establishing antiviral defenses. Upon IFN stimulation, receptor‑associated JAK1 and TYK2 phosphorylate STAT2 (and STAT1). Phosphorylated STAT2 heterodimerizes with STAT1 and associates with IRF9 to form the ISGF3 complex, which binds interferon‑stimulated response elements (ISREs) to induce interferon‑stimulated genes (ISGs), including OAS, PKR (EIF2AK2), and MX1/MX2.
Unlike several other STATs, STAT2 does not bind DNA as a homodimer and exerts most transcriptional functions within ISGF3. In STAT1‑limited contexts, STAT2 can partner with IRF9 to drive a STAT2–IRF9 (ISGF3‑like) response, extending antiviral gene induction.
STAT2 deficiency in humans causes marked susceptibility to viral infections (notably many respiratory RNA viruses, and some DNA viruses), often with severe disease after live‑attenuated vaccines, underscoring STAT2’s non‑redundant role in IFN signaling. Consistently, numerous viruses (e.g., paramyxoviruses, flaviviruses, orthomyxoviruses) encode proteins that degrade, sequester, or block STAT2, highlighting STAT2 as a major target of viral immune evasion.
Because STAT2 tunes IFN pathway amplitude and duration, it represents a potential therapeutic node to enhance antiviral responses or attenuate pathogenic IFN signaling in selected auto‑inflammatory and autoimmune settings.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-STAT2 Tyr690 Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-STAT2 Tyr690 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.
STAT2 activation mediated by Type I interferons
A431 cells were seeded in a 96-well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with increasing concentrations of IFNβ for 30 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT2 Phospho (Tyr690) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 6,000 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, IFNβ triggered a dose-dependent increase in the levels of STAT2 Phospho (Tyr690) with a modest decrease in Total levels (2.8 fold).
Decrease of STAT2 phosphorylation by Ruxolitnib
THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in HBSS containing 10 ng/mL IFNβ at 37°C, 5% CO2 for 15 minutes. The cells were then treated with increasing concentrations of JAK inhibitor, Ruxolitinib for 1 hour.
After treatment, the cells were spun down at 1,200 rpm for 5 minutes and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT2 Phospho (Tyr690) and Total levels were evaluated using respective AlphaLISA SureFire Ultra 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.
Inhibition of JAK1 signaling mediated by Ruxolitinib, resulted in a significant decrease of phosphorylated STAT2 while STAT2 Total levels remained unchanged.
Assay sensitivity – cell lysate dilution
Cell lysate was prepared from A431 cells cultured to confluency in T175 flasks. Cells were treated with 10 ng/mL IFNβ for 30 minutes and then lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and STAT2 Phospho (Tyr690) 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 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. The assay can detect STAT2 Phospho (Tyr690) in less than 200 cells.
