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

ADP² FP ASSAY - LEARN MORE

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

Transcreener ADP² FP Assay Tech Manual (PDF)

ADP² FI ASSAY - LEARN MORE

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

Transcreener ADP² FI Assay Tech Manual (PDF)

ADP² TR-FRET Assay - LEARN MORE

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

Transcreener ADP² TR-FRET Assay Tech Manual (PDF)

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.

Three Fluorescent Readouts Available

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

Fluorescent Intensity

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

TR-FRET

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

Instrument	FP Assays	FI Assays	TR-FRET Assays
TriStar²S LB 942	in review	validated	validated
Mithras² LB 943	in review	validated	validated

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

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

Sense	in review	validated	in review

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

EnVision^®/EnVision^® Xcite	validated	validated	validated

Infinite^® M1000/M1000Pro/Safire2™	validated	validated	validated
Infinite^® M200	not capable	validated	not capable
Infinite^® F500	validated	validated	validated
Infinite^® F200/Ultra^evolution	contact us	validated	contact us
Spark™ 10M	validated	validated	validated

What's Included

Edit
Component	Notes
ADP² Antibody	Antibody specific to the nucleotide ADP. Suggested ADP² antibody concentration for FP 28 μg/mL for 25 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 25 μM for IKK-beta.

What You Will Need

Edit
Component	Notes
IKK-beta Enzyme	We have successfully used recombinant full-length human IKK-beta from ThermoFisher Cat. #*PV3836.* We've used IKK-beta at 7.5 nM for screening purposes.
Enzyme Buffer	20 mM Tris (pH 7.5), 5 mM MgCl_2,, 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 IKKtide at 15 µM (SignalChem Cat. #*133-58*)
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

Transcreener ADP² IKK-beta Assay Kits

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

Edit
Cat. No.	Product Description	Price	Add to Cart
3013-A	Transcreener ADP² FI Assay (200 Assay, 96 Well)	$305.00	Add to Cart
3013-1K	Transcreener ADP² FI Assay (1,000 Assay, 384 Well)	$639.00	Add to Cart
3013-10K	Transcreener ADP² FI Assay (10,000 Assay, 384 Well)	$3,689.00	Add to Cart
3013-100K	Transcreener ADP² FI Assay (10 X 10,000 Assay, 384 Well)	$18,445.00	Add to Cart
3010-1K	Transcreener ADP² FP Assay (1,000 Assay, 384 Well)	$639.00	Add to Cart
3010-10K	Transcreener ADP² FP Assay (10,000 Assay, 384 Well)	$3,689.00	Add to Cart
3010-100K	Transcreener ADP² FP Assay (10 X 10,000 Assay, 384 Well)	$18,445.00	Add to Cart
3011-1K	Transcreener ADP² TR-FRET Red Assay (1,000 Assay, 384 Well)	$699.00	Add to Cart
3011-10K	Transcreener ADP² TR-FRET Red Assay (10,000 Assay, 384 Well)	$3,989.00	Add to Cart
3011-100K	Transcreener ADP² TR-FRET Red Assay (10 X 10,000 Assay, 384 Well)	$19,945.00	Add to Cart

Assay Plates for FI & FP

Cat. No.	Product Description	Price
2233	Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)	$40.00	Add to Cart
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$45.00	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$400.00	Add to Cart

Assay Plates for TR-FRET

Cat. No.	Product Description	Price
2234	Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)	$40.00	Add to Cart
2231	Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)	$50.00	Add to Cart
2232	Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)	$450.00	Add to Cart

IKK 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.

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 k_off 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.

Transcreener® ADP² IKK-beta Assay

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

Discover IKK-beta Inhibitors with the Transcreener ADP² Assay

The Role of IKK-beta As Therapeutic Target

Direct Detection of ADP to Measure IKK Enzymatic Activity

Transcreener ADP² FP Assay Tech Manual (PDF)

Transcreener ADP² FI Assay Tech Manual (PDF)

Transcreener ADP² TR-FRET Assay Tech Manual (PDF)

Download Tutorial

Related Posts

Upcoming Webinar: Targeting Kinases Involved in the Innate Immune Response

IKK Complex, NF-kB, and Inflammation – An Intricate Relationship

JAK3 Inhibitors in the Treatment of Autoimmune Disease

Targeting Adenosine Kinase in the Fight Against Neurological Disorders