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AlphaLISA SureFire Ultra Human and Mouse Phospho-JAK2 (Tyr1008) Detection Kit, 10,000 Assay Points

The AlphaLISA™ SureFire® Ultra™ Human and Mouse Phospho-JAK2 (Tyr1008) assay is a sandwich immunoassay for quantitative detection of phospho-JAK2 (Tyr1008) in cellular lysates using Alpha Technology.

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HELLO10
Feature Specification
Application Cell Signaling
Protocol Time 2h at RT

The AlphaLISA™ SureFire® Ultra™ Human and Mouse Phospho-JAK2 (Tyr1008) assay is a sandwich immunoassay for quantitative detection of phospho-JAK2 (Tyr1008) in cellular lysates using Alpha Technology.

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Save 10% on your first Revvity.com order. Use promo code below.
HELLO10
Product Variants
Unit Size: 100 assay points
Part #:
ALSU-PJAK2-B-HV
List Price
USD 694.00
Unit Size: 500 assay points
Part #:
ALSU-PJAK2-B500
List Price
USD 2,346.00
Unit Size: 10,000 assay points
Part #:
ALSU-PJAK2-B10K
List Price
USD 14,270.00
Unit Size: 50,000 assay points
Part #:
ALSU-PJAK2-B50K
List Price
USD 46,060.00
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).
USD 14,270.00
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Overview

Janus kinase 2 (JAK2) is a crucial component of the JAK-STAT signaling pathway, which governs cell growth, differentiation, and immune responses. JAK2 works in concert with cytokine receptors to activate STAT transcription factors, modulating gene expression. Mutations in JAK2 are associated with hematologic malignancies, including AML, as well as autoimmune diseases like Crohn's disease and ulcerative colitis, which are characterized by severe inflammation.

The AlphaLISA SureFire Ultra Human and Mouse Phospho-JAK2 (Tyr1008) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-JAK2 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
JAK2
Target Class
Phosphoproteins
Target Species
Human
Mouse
Technology
Alpha
Therapeutic Area
Autoimmunity
Oncology
Unit Size
10,000 assay points

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

Induction of JAK2 (Tyr1008) phosphorylation 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 (40,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO2. The cells were starved for 2 hours and then treated with increasing concentrations of IFNγ for 20 minutes.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JAK2 Phospho (Tyr1008) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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, IFNγ triggered a dose-dependent increase in the levels of Phospho JAK2 (Tyr1008) while levels of Total JAK2 remained unchanged.

Primary macrophages treated with IFNg
Induction of JAK2 (Tyr1008) phosphorylation in endogenous cell models

THP-1 cells were seeded in a 96-well plate (100,000 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 washed and starved for 2 hours with HBSS + 0.1% BSA, then treated with increasing concentrations of IFNγ for 20 minutes.

After treatment, the cells were lysed with 60 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JAK2 Phospho (Tyr1008) and Total levels were evaluated using respective AlphaLISA SureFire Ultra 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, IFNγ triggered a dose-dependent increase in the levels of Phospho JAK2 (Tyr1008) while Total JAK2 levels remained unchanged.

THP 1 derived macrophages treated with IFNg

A431 cells were seeded in a 96-well plate (60,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. Cells were treated with increasing concentrations of IFNγ for 15 minutes.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JAK2 Phospho (Tyr1008) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 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.

A431 cells treated with IFNg

RAW 264.7 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 starved for 2 hours and then treated with increasing concentrations of IFNγ for 20 minutes.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JAK2 Phospho (Tyr1008) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 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.

RAW 264.7 cells treated with IFNg
Inhibition of endogenous levels of Phospho JAK2 (Tyr1008) in HEL92.1.7 cells

HEL92.1.7 cells were washed and seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA. The cells were treated with increasing concentrations of Ruxolitinib or Pacritinib for 15 minutes.

After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JAK2 Phospho (Tyr1008) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 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.

JAK2 inhibitors Ruxolitinib and Pacritinib, induced a dose-dependent decrease in the levels of Phospho JAK2 (Tyr1008), while JAK2 Total levels remained unchanged.

HEL92.1.7 cells treated with Ruxolitinib
HEL92.1.7 cells treated with Pacritinib
Inhibition of endogenous levels of Phospho JAK2 (Tyr1008) in THP-1 derived macrophages

THP-1 cells were seeded in a 96-well plate (100,000 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 washed and further stimulated with 100 ng/mL IFNγ for 15 minutes, followed by treatment with increasing concentrations of Ruxolitinib or Pacritinib for 15 minutes. All treatments were performed in HBSS + 0.1% BSA.

After treatment, the cells were lysed with 60 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). JAK2 Phospho (Tyr1008) and Total levels were evaluated using respective AlphaLISA SureFire Ultra 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.

JAK2 inhibitors Ruxolitinib and Pacritinib, induced a dose-dependent decrease in the levels of Phospho JAK2 (Tyr1008), while JAK2 Total levels remained unchanged.

THP 1 derived macrophages treated with Ruxolitinib
THP 1 derived macrophages treated with Pacritinib
Specificity of Phospho JAK2 (Tyr1008) assay

Phospho JAK2 (Tyr1008) levels were assessed in Wild Type (WT) and JAK2 knockout (KO) A549 cells. JAK2 KO cells (Abcam ab267113) and WT A549 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 50 ng/mL IFNγ for 20 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho JAK2 (Tyr1008) levels were evaluated by AlphaLISA SureFire Ultra. 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, JAK2 (Tyr1008) was detected in the WT A549 cells treated with IFNγ but not in the JAK2 KO cell line.

Selectivity of Phospho JAK2 (Tyr1008) Assay

Resources

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