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AlphaLISA SureFire Biotin-Free Human and Mouse Total STAT6 Detection Kit, 10,000 Assay Points
AlphaLISA SureFire Biotin-Free Human and Mouse Total STAT6 Detection Kit, 10,000 Assay Points
AlphaLISA Surefire Ultra Total Protein
| Feature | Specification |
|---|---|
| Application | 細胞シグナル伝達 |
| Protocol Time | 2h at RT |
Product variants
Unit Size: 100 assay points
Part #:
ASBF-TST6-A-HV
Unit Size: 500 assay points
Part #:
ASBF-TST6-A500
Unit Size: 10,000 assay points
Part #:
ASBF-TST6-A10K
Unit Size: 50,000 assay points
Part #:
ASBF-TST6-A50K
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 Human and Mouse Total STAT6 Detection Kit, 10,000 Assay Points
AlphaLISA Surefire Ultra Total Protein
AlphaLISA SureFire Biotin-Free Human and Mouse Total STAT6 Detection Kit, 10,000 Assay Points
Product information
Overview
STAT6 is a transcription factor activated primarily by interleukin-4 (IL-4) signaling, leading to phosphorylation at tyrosine 641. STAT6 dimers regulate genes involved in Th2 immune responses, promoting anti-apoptotic activity and IgE production. Dysregulation of STAT6 is linked to allergic diseases, including asthma and atopic dermatitis, as well as certain cancers, where it supports tumor survival and proliferation.
The AlphaLISA SureFire Biotin-Free Human and Mouse Total STAT6 Detection Kit is a sandwich immunoassay for the quantitative detection of total STAT6 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 protein target in a cell lysate.
The Total-AlphaLISA Surefire assay uses two antibodies which recognize two different distal epitopes 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 a proprietary CaptSure 3 agent to capture one of the detection antibodies, which is labeled with CaptSure 3 tag. In the presence of targeted 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 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 permits the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Total-AlphaLISA SureFire 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
Validation of Phospho (Tyr641)/Total STAT6 in IL-4 treated cells
THP-1 cells were seeded in a 96-well plate (400,000 cells/well) in complete RPMI 1640 medium and then treated with increasing concentrations of IL-4 for 15 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 Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 16,000 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 Biotin-Free
|
| Detection Modality |
Alpha
|
| Product Group |
Kit
|
| Protocol Time |
2h at RT
|
| Shipping Conditions |
Shipped in Blue Ice
|
| Target |
STAT6
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
Mouse
|
| Technology |
Alpha
|
| Therapeutic Area |
Oncology
|
| Unit Size |
10,000 assay points
|
Video gallery
AlphaLISA SureFire Biotin-Free Human and Mouse Total STAT6 Detection Kit, 10,000 Assay Points
AlphaLISA SureFire Biotin-Free Human and Mouse Total STAT6 Detection Kit, 10,000 Assay Points
Resources
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Guide
AlphaLISA SureFire Ultra: the ultimate guide for successful experiments
The definitive guide for setting up a successful AlphaLISA SureFire Ultra assay
Several biological processes are regulated by...
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