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.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The adenosine kinase assay is compatible with 96, 384, and 1536-well formats.
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.
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.
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.
FI Detection Mix (10 μL): 1x Stop & Detect Buffer B, 40 μg/mL ADP Ab-IRDye QC1, 8 nM AlexaFluor 594 Tracer.
TR-FRET Detection Mix (10 μL): 1x Stop & Detect Buffer C, 8 nM ADP Ab-Tb, 1000 nM ADP HiLyte 647 tracer.
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| 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. |
| 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 MgCl2,, 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. |
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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