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

IKK-beta Assay - A Transcreener ADP2 Assay Application

Transcreener ADP² Assay directly measures ADP produced by IKK-beta (IKBKB, inhibitor of kappa light polypeptide gene enhancer in B-cells, or IKK2). These ADP measurements allow researchers to effectively determine the enzyme’s activity. The assay provides a powerful tool to screen compound libraries for IKK-beta modulators to help find new therapies for disease. 

The kit comes complete with the detection reagents required to measure activity. IKK-beta enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

How Does This IKK-beta Assay Work?

The Transcreener IKK-beta Assay determines IKK-beta 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 IKK-beta 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 IKK-beta 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 IKK Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener IKK-beta Assay Schematic

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.

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

ADP TR-FRET Kinase 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

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

Transcreener Mix and Read Assay

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

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. 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

Enzyme Tritation Data IKK-beta Assay

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 Assay Dose Response Curve

IC50 = 0.36

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.

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 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

Supplier Instrument FP Assays
FI Assays
TR-FRET Assays
berthold logo TriStar²S LB 942 in review validated validated
Mithras² LB 943 in review validated validated
bioteklogo Cytation™ 5 validated validated validated
Cytation™ 3 validated validated validated
Cytation™ 1 validated validated validated
Synergy™ H1 validated validated validated
Synergy™ 2/H4/4 validated validated validated
Synergy™ HTX not capable validated not capable
Synergy™ Neo 2 validated validated validated
BMGLABTECH Logo POLARstar® Omega validated validated validated
FLUOstar® Omega not capable validated validated
PHERAstar® FSX validated validated validated
PHERAstar® Plus/FS validated validated validated
CLARIOstar® /Plus validated validated validated
VANTAstar validated validated validated
hidex logo Sense in review validated in review
MDS AT logo Analyst® GT/HT validated validated validated
Gemini® XPS/EM not capable validated not capable
SpectraMax® M2/M2e not capable validated not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable validated validated
SpectraMax® Paradigm validated contact us contact us
SpectraMax® iD3/iD5 in review validated validated
perkinElmerLogo EnVision®/EnVision® Xcite validated validated validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated validated validated
Infinite® M200 not capable validated not capable
Infinite® F500 validated validated validated
Infinite® F200/Ultraevolution contact us validated contact us
Spark™ 10M validated validated validated

What's Included

What You Will Need

Assay Plates for TR-FRET

The Role of IKK-beta As a Therapeutic Target

IKK-beta is a serine kinase in the NF-κB pathway. IKK aids in activiating 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. Because of this, IKK-beta modulators have become emerging targets in drug discovery.

The Transcreener ADP2 IKK-beta Assay is an excellent tool for researchers examining the therapeutic effects of IKK-beta.

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