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AlphaLISA SureFire Ultra Human Phospho-STAT6 (Tyr641) Detection Kit, 50,000 Assay Points
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AlphaLISA SureFire Ultra Human Phospho-STAT6 (Tyr641) Detection Kit, 50,000 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein
| Feature | Specification |
|---|---|
| Application | Cell Signaling |
| Sample Volume | 10 µL |
Product variants
Unit Size: 500 assay points
Part #:
ALSU-PST6-A500
List price
USD 2,392.92
Your online price:
Unit Size: 10,000 assay points
Part #:
ALSU-PST6-A10K
List price
USD 14,400.00
Your online price:
Unit Size: 50,000 assay points
Part #:
ALSU-PST6-A50K
List price
USD 46,000.00
Your online price:
Unit Size: 100 assay points
Part #:
ALSU-PST6-A-HV
List price
USD 708.33
Your online price:
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 Ultra Human Phospho-STAT6 (Tyr641) Detection Kit, 50,000 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein
AlphaLISA SureFire Ultra Human Phospho-STAT6 (Tyr641) Detection Kit, 50,000 Assay Points
Product information
Overview
STAT6 (Signal Transducer and Activator of Transcription 6) is a crucial transcription factor in the JAK/STAT signaling pathway. In humans, STAT6 is encoded by a single gene and plays a central role in various cellular processes, particularly in immune regulation, T helper 2 (Th2) cell differentiation, and allergic responses. Activation of STAT6 occurs through phosphorylation at a key tyrosine residue, Tyr641. This phosphorylation is particularly important for STAT6 dimerization, nuclear translocation, and subsequent transcriptional activity. STAT6 signaling is frequently implicated in various diseases, especially in allergic disorders, asthma, and certain types of cancer, where aberrant activation of the pathway contributes to disease progression, immune dysregulation, and therapy resistance.
The AlphaLISA SureFire Ultra Human phospho-STAT6 (Tyr641) Detection Kit is a sandwich immunoassay designed for the quantitative detection of phosphorylated STAT6 (Tyr641) 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
Alpha SureFire kits can be used for:
- Cellular kinase assays
- Receptor activation studies
- Screening
How it works
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a protein target when phosphorylated at a specific residue.
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the targeted 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 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 permits the cells 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 AlphaLISA SureFire Ultra quality.
Assay validation
Induction of STAT6 phosphorylation in PBMCs stimulated with various cytokines
PBMCs were isolated from healthy donors using Ficoll® Plaque Plus. Cells were seeded in a 96-well plate (400,000 cells/well) and starved for 2 hours in serum-free DMEM. Cells were then treated with the indicated cytokines for 15 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT6 Phospho (Tyr641) levels were evaluated using AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 40,000 cells) were 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, IL-4 was the main activator of STAT6 phosphorylation.
IL-4 induces STAT6 phosphorylation in a dose-dependent manner
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 (20,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO2. Cells were treated with the indicated concentration of IL-4 for 20 minutes.
After treatment, cells were lysed in 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT6 Phospho (Tyr641) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 cells) were 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.
As expected, IL-4 triggered a dose-dependent increase in the levels of Phospho STAT6 (Tyr641) while Total STAT6 levels remained unchanged.
THP-1 cells were seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1 % BSA and treated with increasing concentrations of IL-4 for 20 minutes.
After treatment, the cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT6 Phospho (Tyr641) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 16,000 cells/datapoint) were 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, IL-4 triggered a dose-dependent increase in the levels of STAT6 Phospho Tyr641 while Total STAT6 levels remained unchanged.
HeLa 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 IL-4 for 20 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT6 Total and Phospho (Tyr641) levels were evaluated using respective AlphaLISA SureFire Ultra 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, IL-4 triggered a dose-dependent increase in the levels of Phospho STAT6 (Tyr641) while Total STAT6 levels remained unchanged.HeLa 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 IL-4 for 20 minutes.
After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT6 Total and Phospho (Tyr641) levels were evaluated using respective AlphaLISA SureFire Ultra 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, IL-4 triggered a dose-dependent increase in the levels of Phospho STAT6 (Tyr641) while Total STAT6 levels remained unchanged.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Automation Compatible |
Yes
|
| Brand |
AlphaLISA SureFire Ultra
|
| Cellular or Signaling Pathway |
JAK/STAT
|
| Detection Modality |
Alpha
|
| Lysis Buffer Compatibility |
Lysis Buffer
|
| Molecular Modification |
Phosphorylation
|
| Product Group |
Kit
|
| Sample Volume |
10 µL
|
| Shipping Conditions |
Shipped in Blue Ice
|
| Target |
STAT6
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
|
| Technology |
Alpha
|
| Unit Size |
50,000 assay points
|
Video gallery
AlphaLISA SureFire Ultra Human Phospho-STAT6 (Tyr641) Detection Kit, 50,000 Assay Points
AlphaLISA SureFire Ultra Human Phospho-STAT6 (Tyr641) Detection Kit, 50,000 Assay Points
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Application Note
Characterizing chemokine receptor inhibitors with AlphaLISA SureFire Ultra, Alpha SureFire Ultra Multiplex and LANCE Ultra cAMP assays
The measurement of protein phosphorylation is a useful tool for measuring the modulation of receptor activation by both antibodies...
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