-->

HTRF Human Total JAK1 Detection Kit HTRF®

The Total JAK1 kit is designed to monitor the expression level of cellular JAK1, and can be used as a normalization assay for the Phospho-JAK1 Tyr1034/1035 Detection Kit .

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

The Total JAK1 kit is designed to monitor the expression level of cellular JAK1, and can be used as a normalization assay for the Phospho-JAK1 Tyr1034/1035 Detection Kit .

-
In stock

Overview

JAK1 (Janus kinase 1) belongs to the family of non-receptor Janus tyrosine kinases with JAK2, JAK3, and TYK2. A wide array of cytokines and growth factors ( IL6, IL4, IFNalpha, 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/STATs 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
  • PRECISION

Total JAK1 assay principle

The Total-JAK1 assay quantifies the expression level of JAK1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-JAK1 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 JAK1 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 JAK1  assay

Total-JAK1 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 JAK1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF Total JAK1  assay principle

Total-JAK1 1-plate assay protocol

Detection of total JAK1 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.

Validation of JAK1 kits on HEL92.1.7 cells

Inhibition measured with Phospho-JAK1 (Tyr1034/1035) & Total-JAK1 kits on HEL92.1.7 cells

HEL92.1.7 cells (Human erythroleukaemia) were seeded in a half area 96-well culture-treated plate at 300,000 cells / well in 20 µL complete culture medium. Cells were treated with 5 µL of increasing concentrations of Ruxolitinib or Upadacitinib for 1h at 37 ° C, 5% CO2 followed by a stimulation step with 5 µL of pervanadate 100 µM, IL4 100 ng/mL and IFNg 100 ng/mL mixture during 30 minutes . After treatment, cells were lysed with 10µl of supplemented lysis buffer # 4 (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-JAK1 (Tyr1034/1035) or Total JAK1 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, the results obtained show a dose-response inhibition of JAK1 Y1034/1035 phosphorylation upon treatment with Ruxolitinib or Upadacitinib, while the JAK1 expression level remains constant.

Validation of JAK1 kits on HEL92.1.7 cells
Validation of JAK1 kits on HEL92.1.7 cells

Inhibition of JAK1 phosphorylation (Tyr1034/1035) on Hep-G2 cells

Human HEP-G2 cells (hepatocellular carcinoma) were plated in 96-well culture-treated plate (400,000 cells/well) in complete culture medium, and incubated 6H at 37°C,5%CO2. Cells were treated with a dose-response of Ruxolitinib or Upadacitinib overnight at 37 °C, 5% CO2. Cells were stimulated with pervanadate 100 µM, IL6 100 ng/mL, IL4 100 ng/mL and IFNg 100 ng/mL during 1 hour at 37°C, 5%CO2. Cells were then lysed with 25 µl of supplemented lysis buffer #4 (1X) 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-JAK1 (Tyr1034) or Total-JAK1 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, both inhibitors induced a dose-dependent decrease in JAK1 phosphorylation, without effect on the expression level of the receptor.

Validation of JAK1 kits on Hep-G2 cells
Validation of JAK1 kits on Hep-G2 cells

Total JAK1 down-regulation by siRNA

HEL92.1.7 cells were treated with 2.5 µM of Accell siRNA (Horizon) targeting specifically JAK1 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, the cells were lysed with 50 µL of supplemented lysis buffer #4 (1X) and 16 µL of lysates were transferred into a low volume white microplate before the addition of 4 µL of premixed HTRF Total JAK1 detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.

Cell treatment with JAK1 siRNA led to a significant downregulation of JAK1 as highlighted by the 84% signal decrease compared to the cells transfected with the non-targeting siRNA.

​​​​​​​
siRNA experiments on Total JAK1

HTRF Total JAK1 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 #4 (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-JAK1 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 JAK1 kit with Western Blot

Simplified JAK1 signaling pathway

JAK1 in combination with JAK2, JAK3, or Tyk2 interacts with different types of cytokine/interferon receptors (IL2, IL4, IL6 , IFN alpha and gamma). Upon cytokine activation, JAK1 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.
JAK1 signaling pathway

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Product Insert JAK1 total Kit / 64JAK1TPEG-64JAK1TPEH

64JAK1TPEG-64JAK1TPEH - Product Insert

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

Let's find your reader