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Learn More About Transcreener Method Used for This IRAK4 Assay

IRAK4 Assay - A Transcreener ADP2 Assay Application

Transcreener ADP² Kinase Assay directly measures ADP produced by IRAK4 (interleukin-1-receptor-associated kinase 4). These ADP measurements allow researchers to effectively determine the activity of the IRAK4 enzyme. The assay provides a powerful tool to screen compound libraries for IRAK4 modulators to help find new therapies for disease. 

The kit comes complete with the detection reagents required to measure activity. IRAK4 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme. This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.

Learn how Genentech used the Transcreener ADP² Assay to discover IRAK4 inhibitors.

How Does This IRAK4 Assay Work?

The Transcreener IRAK4 Assay determines IRAK4 enzyme activity by directly measuring the ADP formed by the enzyme using the Transcreener ADP Assay. By detecting ADP output, the 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. 

Direct Detection of ADP to Measure IRAK4 Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener IRAK4 Assay Schematic

The workhorse. Used in many large screens. Best deck and signal stability.

Fluorescent Intensity (FI)

ADP FI Kinase Assay Kit

Positive FI signal. Compatible with simple fluorescence plate readers. Faster read time than FP or TR-FRET.

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

ADP TR-FRET Kinase Assay Kit

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®.

Applications

  • Measure Enzymatic Activity of IRAK4
  • Screen Compound Libraries for IRAK4 Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Measure Drug-Target Residence Time

Features

  • Direct detection of unlabeled ADP
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method
  • Available in FP, FI, or TR-FRET readouts

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

Run your enzyme reaction, add Transcreener reagents, and read your plates. The IRAK4 assay is compatible with 96, 384, and 1536-well formats. 

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized IRAK4 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.

IRAK4 Assay Z prime data

Detection of ADP Under IRAK4 Initial Velocity - FP Readout

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.

Human IRAK4 Enzyme Titration

IRAK4 Assay Enzyme Titration

Linear Response

Linear Response Curve IRAK4 Assay

Screen for Inhibitors & Perform SAR

Transcreener Assays are 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.

Dose-Response - Known IRAK1/4 Inhibitor

IC50 = 0.691 µM

IRAK4 Assay Services

Interested in moving your program forward, but don't want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your IRAK4 program.

Assay Development Services

Lead Discovery Services Include:

  • Inhibitor Screening – To identify or confirm activity with the target.
  • Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC50 results.
  • Residence Time Measurements – Determination of koff using ‘jump dilution’ enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.

Contact Us to Learn More

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

Far-Red FP, FI & TR-FRET Readouts Validated on Major Multimode Readers

SupplierInstrumentFP AssaysFI AssaysTR-FRET Assays
TriStar2S LB 942In ReviewClick Me ArrowValidatedClick Me ArrowValidated
Tristar 5In ReviewClick Me ArrowValidatedIn Review
Mithras2 LB 943In ReviewClick Me ArrowValidatedClick Me ArrowValidated
CytationTM 5ValidatedValidatedValidated
CytationTM 3ValidatedValidatedValidated
CytationTM 1ValidatedValidatedValidated
SynergyTM H1ValidatedValidatedValidated
SynergyTM 2/H4/4Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SynergyTM HTXNot CapableValidatedNot Capable
SynergyTM Neo 2ValidatedValidatedValidated
POLARstar® OmegaClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
FLUOstar® OmegaNot CapableClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SenseIn ReviewClick Me ArrowValidatedIn Review
Analyst® GT/HTClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Gemini® XPS/EMNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M2/M2eNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not CapableClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowValidatedClick Me ArrowContact UsClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidatedIn ReviewClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M200Not CapableClick Me ArrowValidatedNot Capable
Infinite® F500Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact UsClick Me ArrowValidatedClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated

What's Included

What You Will Need

Interconnections Between the DNA Damage Response and Innate Immunity

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. The enzyme targets discussed include:

DDR

  • POLQ
  • WRN
  • PARP1
  • PARG
  • DNA PK

Innate Immunity

  • cGAS
  • TREX1
  • TBK1
  • IKKβ
  • IRAK4
  • ENPP1
  • CD38
  • OAS-1
  • RIG-1/MDA5
  • DDX3
  • CD39
  • CD73
DNA Damage Response and Innate Immunity Preview

The Role of IRAK4 As a Therapeutic Target

As upstream toll-like receptors activate IRAK4, it phosphorylates IRAK1, stimulating downstream substrates by way of its kinase activity. This 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 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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