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

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.

This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.

Application Note - Detection of Adenosine Kinase Activity with the Transcreener ADP2 Kinase Assay

This application note describes methods for using the Transcreener ADP2 Kinase Assay to measure ADK enzymatic activity in a high throughput (384-well) format, e.g., for inhibitor screening and/or inhibitor dose-response measurements.

Application Note Preview Image

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)

Adenosine Kinase Assay Schematic

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

ADP FI Kinase Assay Kit

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 Kit

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

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. 

Transcreener Mix and Read Assay

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.

Adenosine Kinase Z Prime Graph

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

Adenosine Kinase Enzyme Titration v3

Linear Response

Adenosine Kinase Enzyme Titration Linearity v3

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 mM Tris, 0.01% Brij, 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

Adenosine Kinase Enzyme Titration FI Assay v2

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

TR-FRET

Adenosine Kinase Enzyme Titration TR-FRET Assay v3

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.

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

SupplierInstrumentFP AssaysFI AssaysTR-FRET Assays
TriStar2S LB 942In ReviewClick Me ArrowValidatedClick Me ArrowValidated
Tristar 5In ReviewClick Me ArrowValidatedIn Review
Mithras2 LB 943In ReviewClick Me ArrowValidatedClick Me ArrowValidated
CytationTM 5ValidatedValidatedValidated
CytationTM 3ValidatedValidatedValidated
CytationTM 1ValidatedValidatedValidated
SynergyTM H1ValidatedValidatedValidated
SynergyTM 2/H4/4Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SynergyTM HTXNot CapableValidatedNot Capable
SynergyTM Neo 2ValidatedValidatedValidated
POLARstar® OmegaClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
FLUOstar® OmegaNot CapableClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SenseIn ReviewClick Me ArrowValidatedIn Review
Analyst® GT/HTClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Gemini® XPS/EMNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M2/M2eNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not CapableClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowValidatedClick Me ArrowContact UsClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidatedIn ReviewClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M200Not CapableClick Me ArrowValidatedNot Capable
Infinite® F500Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact UsClick Me ArrowValidatedClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated

What's Included

What You Will Need

Assay Plates for TR-FRET

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.

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