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AlphaLISA SureFire Ultra High Performance Human Phospho-STAT6 (Tyr641) Detection Kit, 10,000 Assay Points

AlphaLISA SureFire Ultra Phospho-Protein image
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AlphaLISA SureFire Ultra High Performance Human Phospho-STAT6 (Tyr641) Detection Kit, 10,000 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein image
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AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Phospho-Protein
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Phospho-Protein image
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The AlphaLISA™ SureFire® Ultra™ High Performance Human Phospho-STAT6 (Tyr641) assay is a sandwich immunoassay for quantitative detection of phospho-STAT6 (Tyr641) in cellular lysates using Alpha Technology.

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Feature Specification
Application Cell Signaling
Protocol Time 2h at RT

The AlphaLISA™ SureFire® Ultra™ High Performance Human Phospho-STAT6 (Tyr641) assay is a sandwich immunoassay for quantitative detection of phospho-STAT6 (Tyr641) in cellular lysates using Alpha Technology.

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Product Variants
Unit Size: 100 assay points
Part #:
ALSU-PST6-B-HV
Unit Size: 500 assay points
Part #:
ALSU-PST6-B500
Unit Size: 10,000 assay points
Part #:
ALSU-PST6-B10K
Unit Size: 50,000 assay points
Part #:
ALSU-PST6-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).
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AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Phospho-Protein image
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AlphaLISA SureFire Ultra High Performance Human Phospho-STAT6 (Tyr641) Detection Kit, 10,000 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein image
Video Play Button
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Phospho-Protein
AlphaLISA SureFire Ultra High Performance Human Phospho-STAT6 (Tyr641) Detection Kit, 10,000 Assay Points
AlphaLISA SureFire Ultra Phospho-Protein image
AlphaLISA SureFire Ultra Phospho-Protein image
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Product information

Overview

Signal Transducer and Activator of Transcription 6 (STAT6) is a critical transcription factor in the JAK/STAT signaling pathway, primarily activated by interleukin-4 (IL-4) and interleukin-13 (IL-13). Upon activation, STAT6 dimerizes and translocates to the nucleus, where it regulates the expression of genes involved in anti-apoptotic and T helper 2 (Th2) immune responses. Dysregulation of STAT6 drives inflammation and hypersensitivity in allergic diseases like asthma, supports tumor survival and proliferation in cancers such as lymphomas, and plays a minor role in antiviral immunity through the STING pathway.

The AlphaLISA SureFire Ultra High Performance Human Phospho-STAT6 (Tyr641) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-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 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

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
AlphaLISA SureFire Ultra
Detection Modality
Alpha
Protocol Time
2h at RT
Shipping Conditions
Shipped in Blue Ice
Target
STAT6
Target Class
Phosphoproteins
Target Species
Human
Technology
Alpha
Therapeutic Area
Oncology
Unit Size
10,000 assay points

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AlphaLISA SureFire Ultra High Performance Human Phospho-STAT6 (Tyr641) Detection Kit, 10,000 Assay Points
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AlphaLISA SureFire Ultra High Performance Human Phospho-STAT6 (Tyr641) Detection Kit, 10,000 Assay Points
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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.

Assay Principle Phospho AlphaLISA SureFire Ultra

 

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.

2 Plates Assay Protocol AlphaLISA Surefire Ultra Phospho Assay
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.

1 plate assay protocol AlphaLISA Surefire Ultra Phospho assay

Assay validation

Activation of STAT6 Phospho (Tyr641) 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 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 Phospho (Tyr641) and Total levels were evaluated using AlphaLISA SureFire Ultra Phospho (Tyr641) (HP) and Total STAT6 assays. For the detection step, 10 µL of cell lysate (approximately 3,000 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.

Primary macrophages treated with IL-4
Activation of STAT6 Phospho (Tyr641) in THP-1 cells

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 AlphaLISA SureFire Ultra Phospho (Tyr641) (HP) and Total STAT6 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 (HP) while Total STAT6 levels remained unchanged.

THP-1 cells treated with IL-4
Activation of STAT6 Phospho (Tyr641) in A431 cells

A431 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 then treated for 20 minutes with increasing concentrations of IL-4.

After treatment, the cells were lysed with 150 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT6 Phospho (Tyr641) and Total levels were evaluated using AlphaLISA SureFire Ultra Phospho (Tyr641) (HP) and Total STAT6 assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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 Phospho STAT6 Tyr641 (HP) while Total STAT6 levels remained unchanged.

A431 cells treated with IL-4

Resources

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Guide
AlphaLISA SureFire Ultra: the ultimate guide for successful experiments

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