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HTRF Human Total JAK1 Detection Kit, 500 Assay Points

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.

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 & disposal requirements under European REACH regulations (EC 1907/2006).

Product Variants
Part number: 64JAK1TPEG
Unit Size: 500 Assay Points
List price: USD 2,147.00
Your price:
USD 2,147.00
USD 2,147.00 /each
Part number: 64JAK1TPEH
Unit Size: 10,000 Assay Points
List price: USD 12,490.00
Your price:
USD 0.00
USD 12,490.00 /each


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.


Protein Detection
Assay Points
Assay Target Type
Assay Technology
Automation Compatible
Detection Method
Experimental Type
In vitro
Shipping Conditions
Shipped in Dry Ice
Therapeutic Area
Unit Size
500 Assay Points

Video gallery

How it works

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.



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.



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.



Assay validation

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.

assay-validation-jak1-total-1 (1).svg




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.





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.



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.



Simplified pathway

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.




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