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HTRF Human Total JAK2 Detection Kit HTRF®

The Total JAK2 kit is designed to monitor the expression level of cellular JAK2, and can be used as a normalization assay for the Phospho-JAK2 Tyr1007/1008 Detection Kit .

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  • No-wash No-wash
  • High sensitivity High sensitivity
  • All inclusive kit All inclusive kit
  • Low sample consumption Low sample consumption

The Total JAK2 kit is designed to monitor the expression level of cellular JAK2, and can be used as a normalization assay for the Phospho-JAK2 Tyr1007/1008 Detection Kit .

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Overview

JAK2 (Janus kinase 2) belongs to the family of non-receptor Janus tyrosine kinases with JAK1, JAK3, and TYK2. A wide array of cytokines and growth factors ( IL6, IL4, IFN alpha, GM-CSF) attached to their receptors induce the phosphorylation of JAKs. The activated JAKs subsequently phosphorylate additional targets, including both the cytokine receptors and the major substrates: STATs.  The JAKs/SATs signaling stimulates cell proliferation, differentiation, migration, and apoptosis. Altering JAK/Stat signaling to reduce cytokine induced pro-inflammatory responses represents an attractive target for anti-inflammatory therapies.

Benefits

  • SPECIFICITY
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Total JAK2 assay principle

The Total-JAK2 assay quantifies the expression level of JAK2 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-JAK2 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of JAK2 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Principle of the HTRF Total JAK2 assay

Total-JAK2 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Total JAK2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF Total JAK2 assay

Total-JAK2 1-plate assay protocol

Detection of total JAK2 with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

One-plate protocol of the HTRF Total JAK2 assay

Inhibition measured with Phospho-JAK2 (Tyr1007/1008) & Total-JAK2 kits on HEL92.1.7 cell

HEL92.1.7 cells (Human erythroleukaemia) were seeded in a half area 96-well culture-treated plate at 200,000 cells / well in 20 µL complete culture medium. Cells were treated with 5 µL of increasing concentrations of Ruxolitinib (type I JAK inhibitor) or CHZ868 (Type II JAK inhibitor) for 1h at 37 ° C, 5% CO2 followed by a stimulation step with 5 µL of pervanadate 100 µM during 30 minutes . After treatment, cells were lysed with 10 µl of supplemented lysis buffer # 1 (4X) for 30 min at RT under gentle shaking.

After cell lysis, 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-JAK2 (Tyr1007/1008) or Total JAK2 detection reagents were added. The HTRF signal was recorded after 3 hours at room temperature.

As expected, inhibitors induced a dose-dependent decrease in JAK2 phosphorylation upon treatment with Ruxolitinib or CHZ868, without effect on the expression level of the total protein.

Validation of JAK2 kits on HEL92.1.7 cells
Validation of JAK2 kits on HEL92.1.7 cells

Inhibition of JAK2 phosphorylation (Tyr1007/1008) on THP1 cells

THP1 cells (Human monocytic leukemia ) were seeded in a half area 96-well culture-treated plate at 400,000 cells / well in 20 µL complete culture medium. Cells were treated with 5 µL of increasing concentrations of Ruxolitinib, Tofacitinib or Pacritinib  for 1h at 37 ° C, 5% CO2 followed by a stimulation step with 5 µL of pervanadate at 100 µM during 30 minutes . After treatment, cells were lysed with 10 µl of supplemented lysis buffer # 1 (4X) for 30 min at RT under gentle shaking.

After cell lysis, 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-JAK2 (Tyr1007/1008) or Total JAK2 detection reagents were added. The HTRF signal was recorded after 3 hours at room temperature.

As expected, the results obtained show a dose-response inhibition of JAK2 Y1007/1008 phosphorylation upon treatment with Ruxolitinib, Tofacitinib or Pacritinib, while the JAK2 expression level remains constant.

Validation of JAK2 kits on THP1 cells
Validation of JAK2 kits on THP1 cells
Validation of JAK2 kits on THP1 cells

Total JAK2 down-regulation by siRNA

HEL92.1.7 cells were treated with 2.5 µM of Accell siRNA (Horizon) targeting specifically JAK2 or with a non-targeting siRNA (included as control), in a 96-well plate (200,000 cells/well) under 100 µL. After 48h incubation at 37°C, 50 µL of complete culture medium was added and the cells were incubated for an additional 24h-incubation at 37°C.  After plate centrifugation, cells were lysed with 50 µL of supplemented lysis buffer #1 (1X) and 16 µL of lysates were transferred into a low volume white microplate before the addition of 4 µL of premixed HTRF Total-JAK2 detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.

Cell treatment with JAK2 siRNA led to a significant downregulation of Total JAK2  with 77% signal decrease compared to the cells transfected with the non-targeting siRNA. 

siRNA experiments on Total JAK2

HTRF Total JAK2 assay compared to Western Blot

HEL92.1.7 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After 72h incubation, the cells were lysed with 3 mL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Total-JAK2 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

A side by side comparison of Western Blot and HTRF demonstrates that the HTRF assay is 8-fold more sensitive than the Western Blot, at least under these experimental conditions.
Comparison of HTRF Total JAK2 kit with Western Blot

Simplified JAK2 signaling pathway

JAK2, in combination with JAK1, JAK3, or Tyk2, interacts with different types of cytokine/interferon receptors (IL2, IL4, IL6 , IFN alpha and gamma). Upon cytokine activation, JAK2 and the other members of the JAK family transphosphorylate each other, and then activate the transcription factors STAT1, STAT2, STAT3, STAT5, and STAT6. In turn they dimerize and translocate to the nucleus to trigger the transcription of genes regulating cell differentiation, proliferation, survival, and adapted immune response.

JAK2 signaling pathway

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Plate Reader Requirement

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