Signal Transducer and Activator of Transcription 1 (STAT1) is a pivotal transcription factor within the JAK/STAT signaling pathway, activated by interferons and various cytokines. Upon activation, STAT1 dimerizes and translocates to the nucleus, where it binds to DNA and regulates the expression of genes involved in immune defense and tumor suppression. Dysregulation of STAT1 can promote or inhibit tumor growth, drive chronic inflammation in autoimmune diseases like lupus and rheumatoid arthritis, and contribute to neuronal damage in neurodegenerative diseases.
The AlphaLISA SureFire Ultra High Performance Human and Mouse Phospho-STAT1 (Tyr701) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-STAT1 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.
Activation of Phospho STAT1 (Tyr701) in primary macrophages
PBMCs were isolated from healthy donors and cultured for 6 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (30,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO2. The cells were then treated with increasing concentrations of IFNa for 10 minutes.
After treatment, the cells were lysed with 150 µL of lysis buffer for 10 minutes at RT with shaking (350 rpm). STAT1 Phospho and Total levels were evaluated using AlphaLISA SureFire Ultra Phospho (Tyr701) (HP) and Total STAT1 assays, respectively. Lysates were further diluted 1:5 in Lysis Buffer for Phospho analysis. For the detection step, 10 µL of cell lysate (approximately 400 cells for Phospho and 2,000 cells Total STAT1) 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, IFNα triggered a dose-dependent increase in the levels of Phospho Tyr701 (HP) while Total STAT1 levels remained unchanged.
Activation of Phospho STAT1 (Tyr701) in HEL92.1.7 cells
HEL92.1.7 cells were seeded in a 96-well plate (400,000 cells/well) and starved for 2 hours in HBSS + 0.1 % BSA at 37°C, 5% CO2. The cells were treated with increasing concentrations of IFNα for 10 minutes.
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 and Total levels were evaluated using AlphaLISA SureFire Ultra Phospho (Tyr701) (HP) and Total STAT1 assays, respectively. Lysates were further diluted 1:10 in Lysis Buffer for Phospho analysis. For the detection step, 10 µL of cell lysate (approximately 4,000 cells for Phospho and 40,000 cells for Total STAT1) 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, IFNα triggered a dose-dependent increase in the levels of Phospho Tyr701 (HP) while Total STAT1 levels remained unchanged.
Activation of Phospho STAT1 (Tyr701) in mouse cells
RAW 264.7 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were starved in medium containing 1% FBS for 2 hours, then treated with increasing concentrations of mouse IFNγ for 20 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT1 Phospho and Total levels were evaluated using AlphaLISA SureFire Ultra Phospho (Tyr701) (HP) and Total STAT1 assays, respectively. For the detection step, 10 µL of cell lysate (approximately 4,000 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, IFNγ triggered a dose-dependent increase in the levels of Phospho Tyr701 (HP) while Total STAT1 levels remained unchanged.
Activation of Phospho STAT1 (Tyr701) in THP-1 derived macrophages
THP-1 cells were seeded in a 96-well plate (100,000 cells/well) in complete medium containing 100 nM of PMA for 24 hours at 37°C, 5% CO2. The THP-1 derived macrophages were starved for 2 hours in HBSS + 0.1 % BSA and then treated with increasing concentrations of IFNα or IFNγ for 15 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT1 Phospho and Total levels were evaluated using AlphaLISA SureFire Ultra Phospho (Tyr701) (HP) and Total STAT1 assays, respectively. Lysate was further diluted 1:10 in Lysis Buffer for STAT1 Phospho analysis. For the detection step, 10 µL of lysate (approximately 1,000 cells for Phospho and 10,000 cells for Total STAT1) 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, IFNα and IFNγ triggered a dose-dependent increase in the levels of STAT1 Phospho Tyr701 (HP) while Total STAT1 levels remained unchanged.
