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AlphaLISA SureFire Ultra Human & Mouse Total STAT6 Detection Kit, 50,000 Assay Points

AlphaLISA Surefire Ultra Total Protein
Total AlphaLISA SureFire Ultra Detection Kit
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AlphaLISA SureFire Ultra Human & Mouse Total STAT6 Detection Kit, 50,000 Assay Points
AlphaLISA Surefire Ultra Total Protein
Total AlphaLISA SureFire Ultra Detection Kit
Video Play Button
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AlphaLISA Surefire Ultra Total Protein
Total AlphaLISA SureFire Ultra Detection Kit
AlphaLISA Surefire Ultra Total Protein
AlphaLISA Surefire Ultra Total Protein
Total AlphaLISA SureFire Ultra Detection Kit
AlphaLISA Surefire Ultra Total Protein
Total AlphaLISA SureFire Ultra Detection Kit
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The AlphaLISA™ SureFire® Ultra™ total STAT6 assay is a sandwich immunoassay for quantitative detection of STAT6 (phosphorylated and non-phosphorylated) in cellular lysates using Alpha Technology. This assay is intended to be a normalization phosphorylation studies.

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Feature Specification
Application Cell Signaling
Sample Volume 10 µL
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The AlphaLISA™ SureFire® Ultra™ total STAT6 assay is a sandwich immunoassay for quantitative detection of STAT6 (phosphorylated and non-phosphorylated) in cellular lysates using Alpha Technology. This assay is intended to be a normalization phosphorylation studies.

Click to copy promo code to clipboard.
March lab savings - SAVE 10%. Use promo code below.
MARCH10
Terms and conditions apply.
View product information
Product Variants
Unit Size: 500 assay points
Part #:
ALSU-TST6-A500
List Price
USD 2,392.92
Your online price:
Unit Size: 10,000 assay points
Part #:
ALSU-TST6-A10K
List Price
USD 14,400.00
Your online price:
Unit Size: 50,000 assay points
Part #:
ALSU-TST6-A50K
List Price
USD 46,000.00
Your online price:
Unit Size: 100 assay points
Part #:
ALSU-TST6-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).
Total list price:
USD 46,000.00
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AlphaLISA Surefire Ultra Total Protein
Total AlphaLISA SureFire Ultra Detection Kit
AlphaLISA Surefire Ultra Total Protein
Total AlphaLISA SureFire Ultra Detection Kit
Video Play Button
Video Play Button
AlphaLISA SureFire Ultra Human & Mouse Total STAT6 Detection Kit, 50,000 Assay Points
AlphaLISA Surefire Ultra Total Protein
Total AlphaLISA SureFire Ultra Detection Kit
Video Play Button
Video Play Button
AlphaLISA Surefire Ultra Total Protein
Total AlphaLISA SureFire Ultra Detection Kit
AlphaLISA Surefire Ultra Total Protein
AlphaLISA SureFire Ultra Human & Mouse Total STAT6 Detection Kit, 50,000 Assay Points
AlphaLISA Surefire Ultra Total Protein
Total AlphaLISA SureFire Ultra Detection Kit
AlphaLISA Surefire Ultra Total Protein
Total AlphaLISA SureFire Ultra Detection Kit
Video Play Button
Video Play Button

Product information

Overview

The AlphaLISA SureFire Ultra total STAT6 assay is a sandwich immunoassay for quantitative detection of total STAT6 (phosphorylated and non-phosphorylated) 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.

In the AlphaLISA SureFire Ultra assay, Donor beads are coated with streptavidin to capture one of the antibodies, which is biotinylated. Acceptor beads are coated with a proprietary CaptSure™ agent that immobilizes the other antibody, labeled with a CaptSure™ tag. In the presence of target protein, the two antibodies bring the Donor and Acceptor beads close together, generating signal. The amount of light emission is directly proportional to the amount of protein present in the sample.

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

Specifications

Application
Cell Signaling
Automation Compatible
Yes
Brand
AlphaLISA SureFire Ultra
Detection Modality
Alpha
Lysis Buffer Compatibility
Lysis Buffer
Molecular Modification
Total
Product Group
Kit
Sample Volume
10 µL
Shipping Conditions
Shipped in Blue Ice
Target
STAT6
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
Alpha
Therapeutic Area
Autoimmunity
Immuno-oncology
Inflammation
Unit Size
50,000 assay points

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AlphaLISA SureFire Ultra Human & Mouse Total STAT6 Detection Kit, 50,000 Assay Points
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AlphaLISA SureFire Ultra Human & Mouse Total STAT6 Detection Kit, 50,000 Assay Points
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How it works

Total-AlphaLISA SureFire Ultra assay principle

The Total-AlphaLISA SureFire Ultra assay measures the expression level of a protein target in a cell lysate.

The Total-AlphaLISA SureFire Ultra 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 streptavidin to capture one of the detection antibodies, which is biotinylated. 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.

assay-principle-Total-AlphaLISA-Surefire-Ultra.jpg

 

Total-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 Total-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.

2plates-assay-protocol-AlphaLISA-Surefire-Ultra.jpg
Total-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Total 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.

1plate-assay-protocol-AlphaLISA-Surefire-Ultra.jpg

Assay validation

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.

img-stat6-phosphorylation-in-primary-macrophages-alsu-tst6-a.jpg

 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.

img-stat6-phosphorylation-in-thp-1-cells-alsu-tst6-a.jpg

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.

img-stat6-phosphorylation-in-hela-cells-alsu-tst6-a.jpg

Resources

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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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