HTRF Human Total JAK1 Detection Kit HTRF®

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.

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.

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.

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.

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.

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.

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.