The phospho-STAT1 (Tyr701)/phospho-STAT2 (Tyr690) complex is the activated heterodimeric transcription factor that mediates type I and type III interferon responses critical for antiviral immunity. Upon interferon receptor engagement, JAK1 and TYK2 phosphorylate STAT1 at Tyr701 and STAT2 at Tyr690, promoting heterodimerization through reciprocal SH2 domain interactions. The phosphorylated heterodimer recruits IRF9 to form the ISGF3 complex, which translocates to the nucleus and binds interferon-stimulated response elements (ISREs) to induce expression of antiviral genes including MX1, OAS, PKR, and ISG15. Unlike STAT1 homodimers that bind GAS elements in response to IFN-γ, the STAT1-STAT2 heterodimer drives a broader interferon-stimulated gene program. Deficiencies in either STAT1 or STAT2 cause severe viral susceptibility, while sustained complex activation contributes to autoimmune diseases and interferonopathies. Many viruses encode proteins that degrade or inhibit STAT2 to evade immune responses, highlighting the complex as a critical host defense mechanism.
The AlphaLISA SureFire Ultra Human Phospho-STAT1 (Tyr701)/phospho-STAT2 (Tyr690) Complex Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-STAT1 (Tyr701)/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
AlphaLISA SureFire Ultra phospho protein complex detection assay principle
The AlphaLISA SureFire Ultra complex assay measures cellular protein-protein interactions (PPI) in a biological sample (e.g. cell lysate).
The assay uses two antibodies that specifically recognize the phosphorylated forms of Protein 1 and Protein 2, respectively. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the first assay antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture the second antibody, which is biotinylated. In the presence of the protein complex, 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 PPI present in the sample.
AlphaLISA SureFire Ultra phospho complex detection 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 complex 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.
AlphaLISA SureFire Ultra phospho protein complex detection one-plate assay protocol
Detection of the phospho protein complex 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.
STAT1/STAT2 Phospho Complex activation by Type I Interferons
RPMI 8226 and THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium at 37°C, 5% CO2 and treated with 10 ng/mL IFNα at the indicated time points.
After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho STAT1 (Tyr701)/Phospho STAT2 (Tyr690) Complex and STAT2 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. As expected, treatment with IFNα triggered rapid phosphorylation of the STAT1/STAT2 Complex with no significant change to Total STAT2 levels.
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). STAT1 Phospho (Tyr701)/STAT2 Phospho (Tyr690) Complex and STAT2 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 p-STAT1/p-STAT2 Complex with a modest decrease in Total STAT2 levels (2.8 fold).
Decrease of STAT1/STAT2 Phospho Complex by Ruxolitnib
THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in HBSS containing 10 ng/mL IFNβ for 15 minutes at 37°C, 5% CO2. The cells were then treated with increasing concentrations of JAK inhibitor, Ruxolitinib for 1 hour.
After treatment, the cells were spun down at 1200 rpm for 5 minutes and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT1 Phospho (Tyr701)/STAT2 Phospho (Tyr690) Complex and Total STAT2 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 decrease of phosphorylated STAT1/STAT2 Complex 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 STAT1 Phospho (Tyr701)/STAT2 Phospho (Tyr690) Complex 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 p-STAT1/p-STAT2 complex in less than 1,000 cells.
