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

Transcreener ADP2 Adenosine Kinase Assay

The Transcreener ADP² Assay directly measures the ADP produced by Adenosine Kinase (also known as ADK or AK). These ADP measurements allow researchers to effectively determine the activity of the Adenosine Kinase 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. Adenosine kinase enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

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. The role of ADK 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)

Direct Detection of ADP to Measure ADK Enzymatic Activity

Adenosine Kinase 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.

ADP TR-FRET ATPase Assay

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

The Transcreener ADP² Adenosine Kinase 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 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

Linear Response

Adenosine Kinase Enzyme Titration Linearity

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

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

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

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

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

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

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