ITK (352-620) GST&His Tag Protein, Human

ITK (Interleukin-2-inducible T-cell kinase) is a core member of the TEC family of non-receptor tyrosine kinases, primarily expressed in T cells and NK cells. This 620-amino acid protein contains five functional domains: PH domain, BTK motif, SH3 domain, SH2 domain, and protein kinase domain. ITK plays a critical role in the T cell receptor (TCR) signaling pathway. Upon TCR activation, ITK is recruited to the cell membrane and phosphorylated by LCK, leading to its full activation. Activated ITK subsequently phosphorylates PLCγ1, triggering Ca²⁺ influx and MAPK pathway activation, ultimately regulating cytokine expression and T cell proliferation and differentiation. ITK is particularly crucial for Th2 cell differentiation; ITK knockout mice exhibit impaired Th2 cell differentiation while retaining Th1 cell function, a phenomenon known as Th1 skewing.

Beyond T cells, ITK is also involved in NKT cell development and NK cell function regulation. Regarding disease associations, ITK dysfunction is closely linked to autoimmune diseases, inflammatory disorders, and T-cell lymphomas. Consequently, ITK inhibitors have garnered significant attention in the fields of inflammatory diseases and cancer immunotherapy, with the selective ITK inhibitor soquelitinib demonstrating potential in modulating tumor immunity.

Assay protocol

Principle: The ITK assay is performed using the ADP-Glo Kinase Assay kit which quantifies the amount of ADP produced by the ITK reaction. The ADP-Glo Reagent is added to terminate the kinase reaction and to deplete the remaining ATP, and then the Kinase Detection Reagent is added to convert ADP to ATP and to measure the newly synthesized ATP using luciferase/luciferin reaction.

Materials

1.Kinase assay buffer(5X): 200 mM Tris-HCl, pH 7.4, 100 mM MgCl₂ and 0.5 mg/mL BSA, 250 μM DTT

2.Kinase assay buffer(1X): 40 mM Tris-HCl, pH 7.4, 20 mM MgCl₂, 0.1 mg/mL BSA, 50 μM DTT

3.ITK (352-620) GST&His Tag Protein, Human

4.UA-Glo® Kinase ADP Assay (UA, Catalog # UA070101)

5.Substrate: Myelin basic protein (Sinobiological, Catalog # M42-51N)

6.Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)

7.Plate Reader (PerkinElmer)

Produce

1.Prepare a substrate/ATP mixture as follows (25 μM example).

2. Dilute the ITK to 20 µg/mL, 10 µg/mL and 5 µg/mL in Kinase Assay Buffer (1x) and dispense 3 μL into each well of a 384-well plate.

3. Initiate the reaction by adding 2 μL of the detection system prepared in Step 1 to each well. Include a detection system with 3 μL Kinase Assay Buffer (1x) as Blank. The reaction volume is 5 μL.

4. Incubate the reaction at room temperature (22–25℃) for 40 minutes.

5.Add 5 μL of ADP-Glo Reagent to the completed reaction, mix briefly and incubate for 40 minutes at room temperature (22–25 ℃).

6.Add 10 μL of Detection Reagent and incubate the plate for 30 minutes at room temperature (22–25 ℃).

7.Read at luminescence, respectively in endpoint mode.

8.Calculate specific activity.

• Standard Curve

1.Dilute the ATP and ADP to 25 μM in Kinase Assay Buffer (1x).

2.Mix 25 μΜ ATP and 25 μM ADP to form an ATP+ADP solution provided below and dispense 5 μL into each well of a 384-well plate.

3.Add 5 μL of ADP-Glo Reagent to the completed reaction, mix briefly and incubate for 40 minutes at room temperature (22–25 ℃).

4.Add 10 μL of Detection Reagent and incubate the plate for 30 minutes at room temperature (22–25 ℃).

5.Read at luminescence, respectively in endpoint mode.

6.Detect optical signals and establish conversion curves.