IRAK4 His Tag Protein, Human

IRAK4 (Interleukin-1 Receptor-Associated Kinase 4) is a serine/threonine kinase. Its N-terminal death domain facilitates assembly with MyD88 and IRAK2 into a 6:4:4 helical Myddosome signaling complex, while its C-terminal kinase domain is activated through dimerization and autophosphorylation. A unique tyrosine gatekeeper residue provides a strategic advantage for the development of selective inhibitors. As a central component of the TLR/IL-1R signaling pathway, IRAK4 activates NF-κB and MAPK pathways to regulate innate immune responses. Deficiency in IRAK4 leads to specific susceptibility to pyogenic bacteria (such as Streptococcus pneumoniae) while preserving normal resistance to viral and fungal infections. Clinically, IRAK4 is not only implicated as a causative gene in primary immunodeficiency disorders but also plays a critical role in driving tumor survival in lymphomas harboring MYD88 mutations. Currently, IRAK4-targeted small-molecule inhibitors and PROTAC degraders have advanced into clinical trials for the treatment of rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa, and B-cell lymphomas. These therapeutic agents achieve precise immunomodulation by either blocking kinase activity or eliminating scaffold functions.

Assay protocol

Principle: The IRAK4 assay is performed using the ADP-GloTM Kinase Assay kit which quantifies the amount of ADP produced by the IRAK4 reaction. The ADP-GloTM 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.IRAK4 His Tag Protein, Human

4.ADP-Glo Kinase Assay (Promega, Catalog # V6930)

5.Substrate: Myelin Basic Protein (MBP) (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 IRAK4 to 40 µg/mL, 20 µg/mL and 10 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

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

1.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.

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

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

4.Read at luminescence, respectively in endpoint mode.

5.Detect optical signals and establish conversion curves.

Experimental Method

Experimental Principle: The IRAK4 activity assay is performed using the ADP-Glo™ Kinase Assay kit, which quantifies the amount of ADP produced by the IRAK4 reaction. The specific steps are as follows: First, add ADP-Glo™ Reagent to terminate the kinase reaction and deplete remaining ATP; then add the Kinase Detection Reagent to convert ADP to ATP, and measure the newly synthesized ATP using the luciferase/luciferin reaction system.

Experimental 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.IRAK4 His Tag Protein, Human

4.ADP-Glo Kinase Assay (Promega, Catalog # V6930)

5.Substrate: Myelin Basic Protein (MBP) (Sinobiological, Catalog # M42-51N)

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

7.Plate Reader (PerkinElmer)

Experimental Steps

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

2.Dilute IRAK4 to 40 µg/mL, 20 µg/mL, and 10 µg/mL in Kinase Assay Buffer (1x) and dispense 3 μL into each well of a 384-well plate.

3.Initiate the reaction by adding2 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. Incubate the reaction at room temperature (22–25°C) for 40 minutes.

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

5.Add 10 μL Detection Reagent and incubate the plate for 30 minutes at room temperature (22–25°C).

6.Read chemiluminescence signals respectively in endpoint mode.

7.Calculate specific activity.

Standard Curve

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

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

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

4.Add 10 μL Detection Reagent and incubate the plate for 30 minutes at room temperature (22-25°C).

5.Read chemiluminescence signals respectively in endpoint mode.

6.Detect optical signals and establish conversion curves.