Learn More About Transcreener Method Used for this Adenosine Kinase Assay.

ADK Assay Methods

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Assay Services

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Adenosine Kinase Assay - A Transcreener ADP² Assay Application

Transcreener ADP² Assay directly measures ADP produced by Adenosine Kinase (ADK). These ADP measurements allow researchers to effectively determine the activity of the ADK enzyme. The assay provides a powerful tool to screen compound libraries for ADK modulators to help find new therapies for disease.

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

How Does This Adenosine Kinase Assay Work?

The Transcreener Adenosine Kinase Assay determines ADK enzyme activity by directly measuring 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 Adenosine Kinase 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 adenosine kinase 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 are also extremely amiable to HTS.

Direct Detection of ADP to Measure Adenosine Kinase 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 ADK
Screen Compound Libraries for ADK 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 adenosine kinase assay is compatible with 96, 384, and 1536-well formats.

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Robust Assay Yields Quality Data

Z’ measurements using optimized ADK 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 = 0.80.

Detection of ADP Under ADK Initial Velocity - FP Readout

The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 100 µM ATP with 10 µM adenosine substrate. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The enzyme buffer includes 50 mM Tris pH 7.5, 5 mM MgCl₂, and 0.01% Brij. The Detection mix included 40 μg/mL ADP² antibody, 4 nM ADP Alexa 633 tracer, and 1X Stop & Detect Buffer B in 10 μL. The ADK enzyme reaction took place for 60 minutes at room temperature. The Detection Mix was added and incubated for 60 minutes and read with a CLARIOstar plate reader.

ADK Enzyme Titration

ADK Assay Enzyme Titration With FP Readout

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 μg/mL ADK, 50 nM Tris, 0.01% Brig, 5 mM MgCl₂, 100 μM ATP, 10 μM Adenosine. The Detection Mix consists of 1X Stop & Detect Buffer B, 40 μg/mL ADP Ab, and 4 nM tracer.

Dose-Response - ABT-702

Adenosine Kinase Dose Repsonse Curve with ABT-702

IC₅₀ = 1.32 µM

Three Fluorescent Readouts Available

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

Fluorescent Intensity

FI Detection Mix (10 μL): 1x Stop & Detect Buffer B, 40 μg/mL ADP Ab-IRDye QC1, 8 nM AlexaFluor 594 Tracer.

TR-FRET

TR-FRET Detection Mix (10 μL): 1x Stop & Detect Buffer C, 8 nM ADP Ab-Tb, 1000 nM ADP HiLyte647 tracer.

Adenosine Kinase 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 ADK 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 100 μM ATP (in the Detection Mixture) for FP - 40 μg/mL, TR-FRET - 8 nM, and FI - 40 μg/mL. 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. The suggested concentration for 100 μM ATP (in the Detection Mixture) for FP - 4 nM, TR-FRET - 1000 nM, and FI - 8 nM.
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 100 μM for ADK.

What You Will Need


Component	Notes
Adenosine Kinase Enzyme	We have successfully used recombinant human ADK from NOVOCIB Cat.#E-Nov5. An ADK concentration of 7 μg/mL was used for screening and SAR.
Enzyme Buffer	50 mM Tris (pH 7.5), 5 mM MgCl_2,, and 0.01% Brij-35. Enzyme buffer components may be optimized as required.
ADO Substrate	10 μM of Adenosine BellBrook Labs Cat. #2206
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 plate 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 Adenosine Kinase 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

Edit
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)	$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 Adenosine Kinase As a Therapeutic Target

Adenosine Kinase is a cytosolic enzyme that acts to modulate levels of adenosine (ADO) in the cell. Extracellular ADO works as a messenger to localized tissue in response to damage and has a shielding effect on a wide range of conditions.

ADK's role in regulating ADO makes it a model candidate for potential therapeutics. Intracellular blocking of ADK has been shown to increase extracellular concentrations of ADO. ADK inhibitors have been tested for various therapies including inflammation and neurological disorders. Other enzymes within the purinergic system that aid in the regulation of the ADO cascade include CD39 and CD73. (Jarvis, 2019)

The Transcreener ADP² Adenosine Kinase Assay is an excellent tool for researchers examining the therapeutic effects of ADK.

Adenosine Kinase Assay – Transcreener Application

Learn More About Transcreener Method Used for this Adenosine Kinase Assay.

ADK Assay Methods

Ordering Information

Assay Services

Related Resources

Adenosine Kinase Assay - A Transcreener ADP2 Assay Application

How Does This Adenosine Kinase Assay Work?

Direct Detection of ADP to Measure Adenosine Kinase 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 ADK Initial Velocity - FP Readout

ADK Enzyme Titration

Linear Response

Screen for Inhibitors & Perform SAR

Dose-Response - ABT-702

IC50 = 1.32 µM

Three Fluorescent Readouts Available

Fluorescent Intensity

TR-FRET

Adenosine Kinase 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 Adenosine Kinase Activity Assays

Assay Plates for FI & FP

Assay Plates for TR-FRET

The Role of Adenosine Kinase As a Therapeutic Target

Related Resources

Adenosine Kinase Assay - A Transcreener ADP² Assay Application

IC₅₀ = 1.32 µM

Transcreener ADP² Assay Kits - For Adenosine Kinase Activity Assays