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

IKK-beta Assay Method

Ordering Information

Assay Services

Related Resources

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)

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

Learn More About The FP Assay

Transcreener ADP FP Assay Tech Manual

Fluorescent Intensity (FI)

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

Learn More About The FI Assay

Transcreener ADP FI Assay Tech Manual

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

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

Learn More About The TR-FRET Assay

Transcreener ADP TR-FRET Assay Tech Manual

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.

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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.

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

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

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

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.

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.

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.

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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
VANTAstar^™	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


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


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
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.

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Transcreener ADP² Assay Kits - For IKK-beta Activity Assays

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


Cat. No.	Product Description	Price	Add to Cart
3013-A	Transcreener ADP² FI Assay (200 Assay, 96 Well)	$329.00	Add to Cart
3013-1K	Transcreener ADP² FI Assay (1,000 Assay, 384 Well)	$689.00	Add to Cart
3013-10K	Transcreener ADP² FI Assay (10,000 Assay, 384 Well)	$3,989.00	Add to Cart
3013-100K	Transcreener ADP² FI Assay (10 X 10,000 Assay, 384 Well)	$19,945.00	Add to Cart
3010-1K	Transcreener ADP² FP Assay (1,000 Assay, 384 Well)	$659.00	Add to Cart
3010-10K	Transcreener ADP² FP Assay (10,000 Assay, 384 Well)	$3,789.00	Add to Cart
3010-100K	Transcreener ADP² FP Assay (10 X 10,000 Assay, 384 Well)	$18,945.00	Add to Cart
3011-1K	Transcreener ADP² TR-FRET Red Assay (1,000 Assay, 384 Well)	$719.00	Add to Cart
3011-10K	Transcreener ADP² TR-FRET Red Assay (10,000 Assay, 384 Well)	$4,109.00	Add to Cart
3011-100K	Transcreener ADP² TR-FRET Red Assay (10 X 10,000 Assay, 384 Well)	$20,545.00	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)	$50.00	Add to Cart
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$55.00	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$449.00	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)	$50.00	Add to Cart
2231	Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)	$60.00	Add to Cart
2232	Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)	$499.00	Add to Cart

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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.

IKK-beta Assay – Transcreener Application

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

IKK-beta Assay Method

Ordering Information

Assay Services

Related Resources

IKK-beta Assay - A Transcreener ADP2 Assay Application

How Does This IKK-beta Assay Work?

Direct Detection of ADP to Measure IKK Enzymatic Activity

Fluorescent Polarization (FP)

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

Applications

Features

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

Robust Assay Yields Quality Data

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

Human IKK-Beta Enzyme Titration

Linear Response

Screen for Inhibitors & Perform SAR

Dose-Response - Staurosporine

IC50 = 0.36

Three Fluorescent Readouts Available

Fluorescent Intensity

TR-FRET

IKK-beta Assay Services

Lead Discovery Services Include:

Contact Us to Learn More

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

What's Included

What You Will Need

Transcreener ADP2 Assay Kits - For IKK-beta Activity Assays

Assay Plates for FI & FP

Assay Plates for TR-FRET

The Role of IKK-beta As a Therapeutic Target

Related Resources

IKK-beta Assay - A Transcreener ADP² Assay Application

IC₅₀ = 0.36

Transcreener ADP² Assay Kits - For IKK-beta Activity Assays