The Transcreener IRAK4 Assay determines IRAK4 kinase activity by directly measuring the ADP formed by the enzyme using the Transcreener ADP Kinase Assay. By detecting ADP output, the kinase assay provides a universal method to assess the activity of any ADP-producing enzyme in real-time.
Transcreener IRAK4 Assay technology uses a simple but highly effective method that consists of an antibody selective to ADP over ATP and a far-red fluorescent tracer. ADP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.
The IRAK4 assay is available with FP, FI, and TR-FRET. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The IRAK4 assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized IRAK4 kinase reaction conditions indicate a robust assay. Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Z’ shown here = 0.77.
The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 10 µM ATP with 0.1 µg/µL MBP substrate. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The IRAK4 enzyme reaction took place for 60 minutes at room temperature. The enzymatic reaction was then halted using Stop & Detect Buffer B included in the kit and assay plates were read. Non-productive hydrolysis of ATP occurs in many kinase reactions due to many factors including autophosphorylation and water exploiting the ATP binding pocket of the enzyme.
The Transcreener IRAK4 Assay is designed for screening compound libraries in a high throughput format. Follow-up SAR can also be performed using the assay to determine inhibitor potency with ease. Assay conditions include 7.5 nM IRAK4, 10 µM ATP, 0.1 µg/µL MBP substrate for an enzyme reaction of 60 minutes at room temperature. IRAK1/4 inhibitor from R&D Systems Cat. # 5665.
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| Component | Notes |
|---|---|
| ADP² Antibody | Antibody specific to the nucleotide ADP. Suggested ADP² antibody concentration (in the Detection Mixture) for FP is 40 μg/mL for 10 μM ATP. Yours may vary depending on, enzyme, and buffer composition. |
| ADP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. |
| Stop & Detect Buffer B, 10X | 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35. Stops enzyme reactions requiring Mg²+. |
| ATP and ADP | ATP and ADP are used to create the ATP/ADP standard curve. ATP is used as a phosphate donor to start the reaction. We suggest using a starting ATP concentration of 10 μM for IRAK4. |
| Component | Notes |
|---|---|
| IRAK4 Enzyme | We have successfully used recombinant human IRAK4 from MilliporeSigma Cat. #14-599 and used IRAK4 at 7.5 nM for screening purposes. |
| Enzyme Buffer | 50 mM Tris (pH 7.5), 10 mM MgCl2,, 0.01% Brij-35. Enzyme buffer components may be optimized as required. |
| Substrate | Use the substrate to be phosphorylated for your experiment. Here we used MBP at 0.10 µg/µL (MilliporeSigma Cat. #M1891). |
| Assay Plates | Assay Plates Can Be Purchased Directly Through BellBrook Labs FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 FI - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513 |
| Plate Reader | A multi-detection microplate reader configured to measure the output of the ADP tracer is required. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates. |
| Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem. |
There is extensive crosstalk between the DNA damage response (DDR) pathways and innate immune pathways. Both the individual pathways and the interconnections between them are a focus for exciting new small molecule drug therapeutics that target cancers and debilitating autoimmune disorders. The Transcreener HTS Assay platform accelerates these efforts by providing a robust and easy-to-use biochemical assay to measure activity of key enzymes in the innate immune and DDR pathway.
In this guide, we provide an overview of the DDR and innate immune pathway, and describe Transcreener Assays and Assay Systems for key therapeutic targets.
As upstream toll-like receptors activate IRAK4, it phosphorylates IRAK1, stimulating downstream substrates by way of its kinase activity. This signaling pathway cascade leads to the activation of MAPKs and NF-κB and the production of pro-inflammatory cytokines. Further research supports IRAK4 as an innate immunity target.
In these studies, IRAK knockout mice experienced little to no inflammation when stressed. Patients with IRAK4 mutations have issues with pro-inflammatory cytokine and anti-inflammatory cytokine production. Compounds that regulate IRAK4 kinase activity provide a potential avenue for small molecule drugs to treat inflammatory and immune disorders.
Some diseases associated with IRAK4 include cancer, lupus, inflammatory bowel disease, arthritis, and diabetes. The Transcreener ADP2 Assay is an excellent tool for researchers examining the therapeutic effects of IRAK4.
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