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AlphaLISA SureFire Biotin-Free High Performance Human and Mouse Total STAT3 Detection Kit, 10,000 Assay Points
AlphaLISA SureFire Biotin-Free High Performance Human and Mouse Total STAT3 Detection Kit, 10,000 Assay Points
AlphaLISA SureFire Biotin Free Total Kit Schematic
The AlphaLISA™ SureFire® Biotin-Free High Performance Human and Mouse Total STAT3 assay is a sandwich immunoassay for quantitative detection of total STAT3 in cellular lysates using Alpha Technology.
| Feature | Specification |
|---|---|
| Application | 細胞シグナル伝達 |
| Protocol Time | 2h at RT |
| Sample Volume | 10 µL |
The AlphaLISA™ SureFire® Biotin-Free High Performance Human and Mouse Total STAT3 assay is a sandwich immunoassay for quantitative detection of total STAT3 in cellular lysates using Alpha Technology.
Product variants
Unit Size: 100 Assay Points
Part #:
ASBF-TST3-B-HV
Unit Size: 500 Assay Points
Part #:
ASBF-TST3-B500
Unit Size: 10,000 Assay Points
Part #:
ASBF-TST3-B10K
Unit Size: 50,000 Assay Points
Part #:
ASBF-TST3-B50K
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption and disposal requirements under European REACH regulations (EC 1907/2006).
AlphaLISA SureFire Biotin-Free High Performance Human and Mouse Total STAT3 Detection Kit, 10,000 Assay Points
AlphaLISA SureFire Biotin Free Total Kit Schematic
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Product information
Overview
Signal Transducer and Activator of Transcription 3 (STAT3) is a transcription factor that mediates signaling downstream of cytokines, growth factors, and oncoproteins to regulate cell proliferation, survival, differentiation, and immune function. STAT3 is activated by phosphorylation at Tyr705 by receptor-associated JAKs and Src family kinases, which promotes STAT3 dimerization, nuclear translocation, and binding to STAT3-responsive elements in target gene promoters. STAT3 drives expression of genes encoding anti-apoptotic proteins (BCL-XL, MCL-1, survivin), proliferative factors (cyclin D1, MYC), and angiogenic mediators (VEGF), making it a potent oncogenic transcription factor when constitutively activated. Constitutive STAT3 activation is observed in a broad range of cancers including hepatocellular carcinoma, head and neck squamous cell carcinoma, triple-negative breast cancer, and multiple myeloma, driven by upstream mutations in JAK2, IL-6 receptor amplification, or loss of negative regulators such as SOCS proteins. Beyond its cell-intrinsic oncogenic roles, STAT3 shapes the tumor immune microenvironment by promoting immunosuppressive myeloid cell differentiation and suppressing anti-tumor T cell responses. Multiple STAT3 inhibitors targeting the SH2 domain, DNA-binding domain, and upstream JAK kinases are in clinical development, with JAK inhibitors ruxolitinib and tofacitinib approved for inflammatory and hematologic indications.
The AlphaLISA SureFire Biotin-Free High Performance Human and Mouse Total STAT3 Detection Kit is a sandwich immunoassay for the quantitative detection of total STAT3 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
How it works
Total-AlphaLISA SureFire Biotin Free assay principle
The Total-AlphaLISA SureFire Biotin Free assay measures the expression level of a target protein in a biological sample (e.g. cell lysate).
The Total-AlphaLISA SureFire Biotin Free assay uses two antibodies which recognize two different distal epitopes 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 the CaptSure3 tag. In the presence of target 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 protein present in the sample.
Total-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 Total-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.
Total-AlphaLISA SureFire Biotin Free one-plate assay protocol
Detection of Total 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.
Assay validation
Induction of STAT3 (Tyr705) phosphorylation in THP-1 derived macrophages
THP-1 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium containing 100 nM PMA and incubated for 24 hours at 37°C, 5% CO2. The THP-1 derived macrophages were serum starved in media for 2 hours and then stimulated with increasing concentrations of either IFN-α or IL-6 for 20 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT3 Phospho (Tyr705) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free HP assays. For the detection step, 10 µL of cell lysate (approximately 5,000 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, IFNα and IL-6 treatment resulted in a dose dependent increase in STAT3 (Tyr705) phosphorylation, while STAT3 Total levels remained unchanged.
Inhibition of STAT3 (Tyr705) phosphorylation in A431 cells
A431 cells were seeded in a 96-well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. Cells were treated with increasing concentrations of AG1478 for 2 hours and then stimulated with 100 ng/mL EGF for 15 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT3 Phospho (Tyr705) and Total levels were evaluated using respective AlphaLISA SureFire Biotin Free HP assays. 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, treatment with AG1478 resulted in a dose-dependent decrease in the levels of Phospho STAT3 (Tyr705), while Total levels remained unchanged.
Assay sensitivity
Assay sensitivity - cell lysate dilution
Cell lysate was prepared from A431 cells cultured to confluency in a T175 flask, treated with 2 µg/mL EGF for 10 minutes and lysed in 10 mL of Lysis Buffer for 10 minutes at RT with shaking.
Lysate was serially diluted in Lysis Buffer and STAT3 Phospho (Tyr705) and Total levels were evaluated using the AlphaLISA SureFire Biotin Free HP assays. For the detection step, 10 µL of cell lysate 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.
Approximate number of cells is indicated. The dotted line represents assay background. The STAT3 High Performance assays have a broad dynamic range and can detect Phospho and Total STAT3 levels in less than 100 cells/datapoint.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Automation Compatible |
Yes
|
| Brand |
AlphaLISA SureFire Biotin-Free
|
| Detection Modality |
Alpha
|
| Product Group |
Kit
|
| Protocol Time |
2h at RT
|
| Sample Volume |
10 µL
|
| Shipping Conditions |
Shipped in Blue Ice
|
| Target |
STAT3
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
Mouse
|
| Technology |
Alpha
|
| Therapeutic Area |
Autoimmunity
Oncology
|
| Unit Size |
10,000 Assay Points
|
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