Stimulator of Interferon Genes (STING) is an adaptor protein that mediates innate immune responses to cytosolic DNA. Upon binding of cyclic dinucleotides (CDNs) produced by cGAS in response to DNA sensing, STING undergoes conformational changes and translocates from the ER to the Golgi, initiating TBK1- and IRF3-dependent induction of type I interferons and inflammatory cytokines. STING plays critical roles in antiviral defense, cancer immunosurveillance, and autoimmune pathogenesis. STING agonists are under investigation for cancer immunotherapy, aiming to boost antitumor immunity, while inhibitors are being explored for autoinflammatory and autoimmune diseases driven by excessive type I IFN signaling.
The AlphaLISA SureFire Ultra Mouse Phospho-STING (Ser365) is a sandwich immunoassay for the quantitative detection of phospho-STING (Ser365) 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.
DMXAA-mediated STING phosphorylation
RAW 264.7 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 20 µg/mL DMXAA mouse STING ligand for the indicated time points.
After treatment, the cells were lysed with 50 µL of Lysis Buffer B for 20 minutes at RT with shaking (350 rpm). STING Phospho (Ser365)* and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 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, DMXAA strongly induced STING phosphorylation leading to a 500-fold increase within an hour. A 2.7-fold decrease in total levels was observed around 4 hours post-treatment.
*The highly specific and sensitive Phospho STING monoclonal antibody used in this assay was developed by Cell Signaling Technology (clone D1C4T, #51865).
DMXAA induces STING phosphorylation in a dose-dependent manner
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 treated with increasing concentrations of DMXAA mouse STING ligand for 1 hour.
After treatment, the cells were lysed with 50 µL of Lysis Buffer B for 20 minutes at RT with shaking (350 rpm). STING Phospho (Ser365) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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, DMXAA induced STING phosphorylation in a dose-dependent manner while no significant changes were observed in total levels 1 hour post-treatment.
Poly dA/dT induces STING phosphorylation
RAW 264.7 cells were seeded in T25 flasks (2 x 106 cells/flask) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were left untreated or transfected with 25 µg/mL Poly dA/dT for 6 hours.
After treatment, the cells were lysed with 1 mL of Lysis Buffer B for 20 minutes at RT with shaking (350 rpm). STING Phospho (Ser365) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 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, Poly dA/dT induced STING phosphorylation leading to a 17-fold increase while total levels did not change significantly.
2’3’ cGAMP induces STING phosphorylation
RAW 264.7 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 100 µg/mL 2’3’ cGAMP for the indicated time points.
After treatment, the cells were lysed with 50 µL of Lysis Buffer B for 20 minutes at RT with shaking (350 rpm). STING Phospho (Ser365) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 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, 2’3’ cGAMP triggered an increase in the levels of mouse Phospho STING peaking at 2 hours. A significant decrease in Total STING levels was observed post-phosphorylation (4- to 11-fold), likely due to its degradation.
