Learn More About Transcreener Method Used for This IKK-beta Assay

Discover IKK-beta Inhibitors with the Transcreener ADP2 Assay

The Transcreener ADP² Kinase Assay directly measures the ADP produced by  IKK-beta (also known as IKBKB, inhibitor of kappa light polypeptide gene enhancer in B-cells, IKK-β, or IKK2). By measuring the production of ADP researchers can effectively determine the activity of the IKK-beta enzyme. The assay provides a powerful tool to screen entire compound libraries for IKK-beta modulators to help find new therapeutics for autoimmune diseases and cancer.

The Role of IKK-beta As Therapeutic Target

IKK-beta is a serine kinase in the NF-κB pathway. IKK aids in the activation of NF-κB by phosphorylating a bound inhibitor IκBα, allowing NF-κB to enter the nucleus, in turn, activating various inflammatory responses. The signaling pathway is part of the innate immune response to bacterial and viral material, among other stimuli. Incorrect regulation can lead to a number of conditions including inflammatory autoimmune disorders and cancer.

Direct Detection of ADP to Measure IKK Enzymatic Activity

ADP FP Kinase Assay

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

ADP FI Kinase Assay

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

ADP TR-FRET ATPase Assay

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

Applications

  • Measure Enzymatic Activity of IKK-beta
  • Screen Compound Libraries for IKK-beta Inhibitors
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling

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

The Transcreener ADP² IKK Assay is in a simple, HTS-ready, mix-and-read format. Run your enzyme reaction, add Transcreener reagents, and read your plates. The assay is compatible with 96, 384, and 1536-well formats.

Detection of ADP Under IKK-Beta Initial Velocity - FP Readout

The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use both 25 µM ATP with 15 µM IKKTide peptide substrates. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. 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 IKK-Beta Enzyme Titration

Linear Response

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 IKK beta, 10 µM ATP, 10 µM IKKtide for an enzyme reaction of 60 minutes at room temperature.

Dose-Response - Staurosporine

IKK Beta Dose Response With Staurosporine

IC50 = 0.36

Robust Assay Yields Quality Data

Z’ measurements using optimized IKK-beta 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 = 0.87.

Z prime of IKK HTS Assay

Three Fluorescent Readouts Available

Choose the readout that is the best fit for your lab based on preference and plate reader compatibility. 

Fluorescent Intensity

IKK Titration FI Readout

Assay conditions for the IKK-beta titration shown include 25 µM ATP, 15 µM IKKtide, and 24 µg/mL ADP² antibody.

TR-FRET

IKK Titration TR-FRET Readout

Assay conditions for the IKK-beta titration shown include 25 µM ATP, 15 µM IKKtide,  55.3 nM tracer, and 8 nM ADP² Tb Antibody.

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

What's Included

What You Will Need

IKK-beta 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 IKK-beta 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

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In addition to our ADP² Assay, Bellbrook Labs also offers the following products to aid in the drug discovery process:

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