The Transcreener ADP Assay determines ATPase enzyme activity by directly measuring the ADP formed by the enzyme during ATP hydrolysis. By detecting ADP output, the assay provides a universal method to assess the activity of any ADP-producing enzyme in real-time.
Transcreener ATPase 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 ATPase 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 also makes it extremely amiable to HTS.
Use this ATPase activity assay kit to:
Many assays are endpoint-only. Transcreener ADP² Kits can be used in kinetic mode, enabling applications that endpoint assays can’t support.
With this kinetic mode capability, you can measure the residence times of inhibitors with a simple biochemical assay to pick leads with the greatest potential to become drugs.
Learn about measuring drug-target residence times in this free article: Guide to Measuring Drug-Target Residence Times with Biochemical Assays.
The universal nature of Transcreener ATPase Assay allows for the screening of virtually any ATPase enzyme. Universal assays also provide an effective technique for difficult targets because you’re measuring ADP directly. Get ahead of the curve with assays ready for emerging targets.
Examples include:
Direct detection of ADP reduces the degree of interference seen with coupling enzyme-based assays, lowering the number of false positives.
Far-red tracers eliminate fluorescent compound interference. This helps you spend less time triaging false hits and more time finding druggable leads.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The ATPase assay kit is compatible with 96, 384, and 1536-well formats.
The high affinity of the ADP antibody generates sensitive and robust detection of ADP down to 10 nM with Z’ values of 0.7 or greater. That means you can run a high-quality ATPase assay using less ATPase enzyme per well, conserving valuable targets.
The ADP FP Assay window at 10% conversion (0.5 µM ADP). The recommended enzyme concentration was an EC80 of 20 ng/mL. The assay demonstrated a Z’ of 0.87 and a lower limit 0f detection at 0.06 μM ADP under these conditions.
The ADP FP Assay window at 10% conversion (0.5 µM ADP). The recommended enzyme concentration was an EC80 of 20 ng/mL. The assay demonstrated a Z’ of 0.87 and a lower limit 0f detection at 0.06 μM ADP under these conditions.
Reagents are stable at room temperature for a minimum of 8 hours. The fluorescent signal stability of Transcreener ADP² Assays lasts at least 24 hours. This flexibility supports large, automated screens without sacrificing assay quality.
Easily detects less than 10% ATP conversion using 0.1 to 1,000 µM ATP. This broad range ensures accurate inhibitor potencies while minimizing enzyme and substrate costs.
ADP/ATP standard curves for a broad range of initial ATP concentrations (0.1 μM to 1000 μM ATP) using the FP, TR-FRET and FI assay respectively. ADP was increased as total ATP + ADP was held constant. Antibody was adjusted based on a linear relationship between EC85 and initial ATP. All assays were performed in 384-well plates (n=24) and read on the Tecan Safire 2 (FP), BMG Labtech PHERAstar Plus (TR-FRET), or the Perkin Elmer EnVision (FI) multimode plate readers.
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 ATPase program.
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Each ATPase activity assay kit includes the detection system required to quantify ADP formation:
| Component | Notes |
|---|---|
| ADP Antibody | Antibody specific to the nucleotide ADP. |
| ADP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. |
| Stop & Detect Buffer | The Stop & Detect Buffer components will stop enzyme reactions that require Mg2+. To ensure that the enzyme reaction is stopped completely, confirm that the EDTA concentration is at least equimolar to the magnesium ion concentration in the reaction. |
| ATP | The ATP supplied in this kit can be used for the enzyme reaction and to create an ADP/ATP standard curve, if desired. |
| ADP | ADP is used to create the ADP/ATP standard curve. |
To run your ATPase assay successfully, you’ll supply:
| Component | Notes |
|---|---|
| Enzyme | Please use purified enzymes for the best results. |
| Enzyme Buffer | User-supplied enzyme buffer components include enzyme, buffer, acceptor substrate, MgCl2 or MnCl2, Brij-35, and test compounds. EGTA is also included in TR-FRET kits. |
| Assay Plates | Assay Plates Can Be Purchased Separately From 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) and Corning 96-well, half-area plates (Cat. # 3686). 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 output of the ADP tracer is required. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense a minimum volume of 0.5 µL into 384-well plates. |
| Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substances and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem. |
ATPases are large macromolecular biological catalyst enzymes that catalyze the hydrolysis from ATP to ADP. As the enzyme hydrolyzes ATP, it yields a free phosphate ion, ADP, and energy. Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a principal role in a wide variety of cellular functions.
ATPase inhibitors are attractive molecular targets for drug discovery. A well-known ATPase inhibitor is the popular pharmaceutical, omeprazole. Omeprazole targets the H+/K+-ATPase system by using selective and irreversible inhibition of the proton pump in the gastric parietal cells. They are also targeted for other areas of health and disease because of their wide variety of cellular functions.
Finding therapeutic methods to reign in overactivity requires selective technologies that can help assess the ability to interact with the target. Robust, HTS-ready assays that are accurate yet flexible provide a powerful tool to accelerate discovery.
Find the right resources to help you get to your solution faster.
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.
What is the Transcreener ADP² ATPase Assay?
The Transcreener ADP² Assay is a homogeneous, mix-and-read format that directly measures ADP produced by ATP hydrolysis rather than relying on coupling enzymes. This enables robust, high-throughput screening (HTS) compatible assays for ATPase enzymes. Note: the ADP assay can be used for any ADP-producing enzyme, including ATPases and Kinases.
Which detection formats are available for the assay?
The assay is available in three detection modes: fluorescence polarization (FP), fluorescence intensity (FI), and TR-FRET. You can choose the mode depending on your instrument capability and screening throughput.
What makes this kit HTS-ready and suitable for screening large compound libraries?
Key features include:
Does the kit include the ATPase enzyme?
No — the ATPase enzyme is not included in the inhibitor screening assay kit. You’ll need to supply your purified ATPase enzyme. If you require help sourcing enzyme, please contact BellBrook Labs.
How does the assay principle work?
The assay detects the amount of ADP produced when ATP is hydrolyzed by an ATPase enzyme. An antibody selective for ADP over ATP binds a far-red fluorescent tracer. As ADP accumulates, it competes with the tracer, changing the fluorescence signal, which is then measured. This enables the determination of enzyme activity.
Can this assay be run in kinetic (real-time) mode as well as endpoint mode?
Yes — the Transcreener ADP² ATPase Assay kits can be used in kinetic mode, allowing measurement of enzyme activity over time or drug-target residence time, in addition to standard endpoint mode.
What are typical assay performance metrics?
Under recommended conditions, the assay has demonstrated Z’ values greater than 0.7. For example, ADP detection down to ~10 nM and an assay window with 0.5 µM ADP at 10% ATP conversion using low enzyme concentrations.
What plate types and instrument configurations are recommended?
For best performance:
What types of research or screening applications is this kit suitable for?
Applications include:
How universal is this assay for different ATPase enzymes?
The assay is designed to be highly universal, since it detects ADP output rather than relying on enzyme‐specific coupling steps. It supports a broad ATP range (0.1 µM to 1000 µM) and is compatible with many ATPase enzyme classes, including motor proteins, helicases, AAA⁺ ATPases, and other emerging targets.
What advantages does this method have compared to traditional ATPase assays?
Advantages include:
Is prototyping or full-service assay development available?
Yes — BellBrook Labs offers ATPase profiling and assay development services. Their scientists can help set up assays, optimize enzyme concentrations, choose detection modes, and profile inhibitors for potency, selectivity and kinetic properties.
BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com
