Transcreener AMP² Assay directly measures AMP produced by CD39 (Cluster of Differentiation 39, Ectonucleoside triphosphate diphosphohydrolase-1, or NTPDase1). These AMP measurements allow researchers to effectively determine the CD39 enzyme activity. The assay provides a powerful tool to screen compound libraries for CD39 modulators to help find new therapies for disease.
The kit comes complete with the detection reagents required to measure activity. CD39 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.
Ectonucleotidases are plasma membrane-bound enzymes with externally oriented active sites that metabolize nucleotides to nucleosides and are crucial for maintaining immune homeostasis. CD39 hydrolyzes ATP and ADP to AMP. AMP is further processed to adenosine leading to a significant impact on many disease states.
The Transcreener CD39 Assay determines CD39 enzyme activity by directly measuring the AMP formed by the enzyme using the Transcreener AMP Assay. By detecting AMP output, the assay provides a universal method to assess the activity of any AMP-producing enzyme in real-time.
Transcreener CD39 Assay technology uses a simple but highly effective method that consists of an antibody selective to AMP and a far-red fluorescent tracer. AMP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.
The CD39 assay is available with FP 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.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The CD39 assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized CD39 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. Both ATP and ADP produce excellent results.
The assay demonstrates linearity when raw data is converted to AMP using a standard curve. CD39 can use a variety of substrates. Here both ATP and ADP are shown under initial velocity conditions.
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 CD39 program.
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| Component | Notes |
|---|---|
| AMP Antibody | Antibody specific to the nucleotide AMP. |
| AMP Tracer | When displaced, changes in fluorescence can be read in an FP or TR-FRET readout, depending on the assay format. |
| Tris Solution | Used to buffer the detection mixture. |
| AMP | AMP is used to create the ATP/AMP or ADP/AMP standard curve. |
| Component | Notes |
|---|---|
| CD39 Enzyme | We have successfully used recombinant human CD39 from R&D Systems Cat. #4397-EN |
| Enzyme Buffer | We have had success with a 50 mM Tris, 10 mM CaCl2, and 0.01% Brij-35 buffer at pH 7.5. |
| ATP or ADP | Use the nucleotide required for your experiment. It is possible to purchase nucleotides direct from BellBrook Labs. |
| 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 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 AMP 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. |
There is extensive crosstalk between the DNA damage response (DDR) pathways and innate immune pathways. Both the individual pathways and the interconnections between them are a focus for exciting new small molecule drug therapeutics that target cancers and debilitating autoimmune disorders. The Transcreener HTS Assay platform accelerates these efforts by providing a robust and easy-to-use biochemical assay to measure activity of key enzymes in the innate immune and DDR pathway.
In this guide, we provide an overview of the DDR and innate immune pathway, and describe Transcreener Assays and Assay Systems for key therapeutic targets.
CD39 is important in Purinergic Signalling, hydrolyzing ATP & ADP to AMP. CD73 completes this pathway by processing AMP into extracellular adenosine. Extracellular adenosine aids tumor growth and metastasis. Inhibiting this pathway shows potential for cancer treatment. CD39 inhibitors also show potential for use in combination with chemotherapy (Guo et al, 2022).
Studies show a key role for adenosine in immunosuppression in the tumor microenvironment, and ectonucleotidases are emerging as promising immuno-oncology targets. The Transcreener AMP2 CD39 Assay is an excellent tool for researchers examining the therapeutic effects of CD39.
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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.
BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com
