Oct 29, 2025
Transcreener® ADP2 TR-FRET Red Assay Technical Manual
- info 1
- 1BellBrook Labs LLC
- BellBrook LabsTech. support phone: +1 (608) 443-2400 email: [email protected]
Protocol Citation: info 2025. Transcreener® ADP2 TR-FRET Red Assay Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly42bqlx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 13, 2025
Last Modified: October 29, 2025
Protocol Integer ID: 229695
Keywords: assay development for new hts target, assay development, inhibitor profiling, selective for adp, inhibitor profiling across multiple target family, assay, atpase, throughput screening, enzyme, adp, carbohydrate kinase, other substrate, new hts target, examples of enzyme, atp, glutamine synthetase
Abstract
The Transcreener® ADP2 TR-FRET Red Assay is a competitive immunoassay for ADP with a far-red, time-resolved Förster-resonance-energy-transfer (TR-FRET) readout. Because it is highly selective for ADP, the assay can be used with any enzyme that converts ATP to ADP, regardless of what other substrates are used. Examples of enzymes include protein, lipid, and carbohydrate kinases, ATPases, DNA helicases, carboxylases, and glutamine synthetase.
The Transcreener® assay is designed specifically for high-throughput screening (HTS), with a single-addition, mix-and-read format. It offers reagent stability and compatibility with commonly used multimode plate readers. The generic nature of the Transcreener® HTS assay platform eliminates delays involved in assay development for new HTS targets and greatly simplifies compound and inhibitor profiling across multiple target families.
Troubleshooting
Introduction
The Transcreener® ADP2 TR-FRET Red Assay is a competitive immunoassay for ADP with a far-red, time-resolved Förster-resonance-energy-transfer (TR-FRET) readout. Because it is highly selective for ADP, the assay can be used with any enzyme that converts ATP to ADP, regardless of what other substrates are used. Examples of enzymes include protein, lipid, and carbohydrate kinases, ATPases, DNA helicases, carboxylases, and glutamine synthetase.
The Transcreener® assay is designed specifically for high-throughput screening (HTS), with a single-addition, mix-and-read format. It offers reagent stability and compatibility with commonly used multimode plate readers. The generic nature of the Transcreener® HTS assay platform eliminates delays involved in assay development for new HTS targets and greatly simplifies compound and inhibitor profiling across multiple target families.
The Transcreener® ADP2 TR-FRET Red Assay provides the following benefits:
- Accommodates ATP concentrations ranging from 0.1 µM to 1,000 µM.
- Excellent data quality (Z’ ≥ 0.7) at low substrate conversion (typically 10% or less).
- Overcomes the need for time-consuming, one-off assay development for individual members within a group transfer enzyme family by using a single set of assay reagents that detect an invariant product.
- Time-gated detection method largely eliminates interference that can result from promptfluorescence of test compounds.
- Far-red tracer further minimizes interference from fluorescent compounds and light scattering.
Figure 1. Schematic overview of the Transcreener® ADP2 TR-FRET Red Assay. The Transcreener® ADP Detection Mixture contains an ADP HiLyte647 tracer bound to an ADP2 antibody conjugated to terbium (Tb). Excitation of the Tb complex in the UV range (~330 nm) results in energy transfer to the tracer and emission at a higher wavelength (665nm) after a time delay. ADP produced by the target enzyme displaces the tracer, which causes a decrease in TR-FRET.
Product Specifications
| Product | Quantity | Part # | |
| Transcreener® ADP2 TR-FRET Red Assay | 1,000 assays* | 3011-1K | |
| 10,000 assays* | 3011-10K |
*The exact number of assays depends on enzyme reaction conditions. The kits are designed for use with 384-well plates, using 20 µL reaction volumes.
IMPORTANT: Antibody centrifugation is required to remove aggregates that can disrupt data quality. Antibodies should be centrifuged at 10,000 x g for 10 minutes before use. Following centrifugation, pipet the solution needed from the top of the aliquot to ensure precipitate is not present in the detection reagents.
Storage
Store all reagents at –80°C upon receipt.
Materials Provided
| Component | Composition | Notes | |
| ADP HiLyte647 Tracer | 10 µM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35 | The concentration of ADP HiLyte647 Tracer needed for an enzyme target depends upon the ATP concentration and buffer conditions in the enzyme reaction (see Section 4.2). Sufficient tracer is included in the kit to complete 1,000 assays (Part # 3011-1K) or 10,000 assays (Part # 3011-10K) at an ATP concentration up to 100 µM ATP. | |
| ADP2 Antibody-Terbium Conjugate | 800 nM solution in HEPES-buffered saline | The final antibody concentration in the reaction is 4 nM in a 20 µL final reaction volume. | |
| Stop & Detect Buffer C, 10X | 500 mM HEPES (pH 7.5), 200 mM EDTA, and 0.2% Brij-35 | The Stop & Detect Buffer C 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. The final concentration of Stop & Detect Buffer C at the time of FRET measurement is 0.5X. | |
| ATP | 5 mM | The ATP supplied in this kit can be used for the enzyme reaction and to create an ADP/ATP standard curve, if desired. | |
| ADP | 5 mM | ADP is used to create the ADP/ATP standard curve |
Note
Caution: ATP is a common reagent in many laboratories; however, it is imperative that a highly purified preparation be used for the Transcreener® assay. If the ATP stock contains impurities, such as ADP, the assay window will be compromised.
Materials Required but Not Provided
- 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.
- Enzyme—Transcreener® ADP2 assays are designed for use with purified enzyme preparations. Contaminating enzymes, such as phosphatases or nucleotidases, can produce background signal and reduce the assay window.
- Enzyme Buffer Components—User-supplied enzyme buffer components include enzyme, buffer, acceptor substrate, MgCl2 or MnCl2, EGTA, Brij-35, and test compounds.
- Plate Reader—A microplate reader configured to measure TR-FRET of the Tb:HiLyte647 donor:acceptor pair is required. This assay has been designed to provide high-quality data on any HTS-qualified instrument configured to measure TR-FRET using standard europium or terbium complexes with emission wavelengths at 615 nm and 665 nm. Validation was completed using PHERAstar Plus Ex337/Em620/Em665 (BMG LABTECH) and Envision Ex320/ Em615/Em665 (Perkin Elmer).
- Assay Plates—It is important to use assay plates that are entirely white with a nonbinding surface. We recommend Corning® 384-well plates (Cat. # 4513).
- Liquid Handling Devices—Use liquid handling devices that can accurately dispense a minimum volume of 2.5 µL into 384-well plates.
Note
Note: Contact BellBrook Labs Technical Service for suppliers and catalog numbers for buffer components, and additional information regarding setup of TR-FRET instruments.
Before You Begin
1. Read the entire protocol and note any reagents or equipment needed (see Section 2.2).
2. Check the TR-FRET instrument and verify that it is compatible with the assay being performed (see Section 4.1).
Protocol
The Transcreener® ADP2 TR-FRET Red Assay protocol consists of 4 steps (Figure 2). The protocol was developed for a 384-well format, using a 10 µL enzyme reaction and 20 µL final volume at the time that the plates are read. The use of different densities or reaction volumes will require changes in reagent quantities.
Figure 2. An outline of the procedure. The assay consists of 4 main steps with a mix-and-read format.
Set Up the Instrument
Becoming familiar with ideal instrument settings for TR-FRET is essential to the success of the Transcreener® ADP2 TR-FRET Red Assay.
4.1.1 Verify That the Instrument Measures TR-FRET
Ensure that the instrument is capable of measuring TR-FRET (not simply fluorescence intensity) of the terbium:HiLyte647 TR-FRET pair (Ex320/Em615/Em665).
Note
Note: A complete list of instruments and instrument-specific application notes can be found online at: https://www.bellbrooklabs.com/technicalresources/instrument-compatibility
Contact BellBrook Labs Technical Service if you have questions about settings and filter sets for a specific instrument.
4.1.2 Define the Maximum TR-FRET Window for the Instrument
Measuring high (0% ATP conversion) and low (100% ATP conversion) FRET will define the maximum assay window of your specific instrument. Prepare High and Low FRET Mixtures in quantities sufficient to perform at least 6 replicates for each condition.
Use ATP and ADP HiLyte647 Tracer at 0.5X concentration in a 20 µL final reaction volume. This mimics the 2-fold dilution when adding an equal volume of detection mixture to an enzyme reaction. As an example, the 1X detection mixture may contain 10 µM ATP. After adding this to the enzyme reaction, the concentration in the final 20 µL reaction volume would be 5 µM.
High FRET Mixture
Prepare the following solution:
| Component | Stock Concentration | Final Concentration | Example: 25 Assays | Your Numbers | |
| ADP2 Antibody-Tb | 800 nM | 4 nM | 2.5 µL | ||
| 10X Stop & Detect Buffer C | 10X | 0.5X | 25.0 µL | ||
| ADP HiLyte647 Tracer | 10 µM | 13.4 nM | 0.7 µL | ||
| ATP | 5 mM | 5 µM | 0.5 µL | ||
| Water | 471.3 µL | ||||
| Total | 500.0 µL |
The assay window will depend upon your initial ATP concentration. These volumes can be adjusted for fewer assays and different ATP concentrations.
Low FRET Mixture
Prepare the following solution:
| Component | Stock Concentration | Final Concentration | Example: 25 Assays | Your Numbers | |
| ADP2 Antibody-Tb | 800 nM | 4 mM | 2.5 µL | ||
| 10X Stop & Detect Buffer C | 10X | 0.5X | 25.0 µL | ||
| ADP HiLyte647 Tracer | 10 µM | 13.4 nM | 0.7 µL | ||
| ADP | 5 mM | 5 µM | 0.5 µL | ||
| Water | 471.3 µL | ||||
| Total | 500.0 µL |
The assay window will depend upon your initial ADP concentration. These volumes can be adjusted for fewer assays and different ADP concentrations.
Measure the TR-FRET
Test the Z’ factor and assay window on your instrument by adding 20 µL of the Low FRET Mixture in 16 wells and 20 µL of High FRET Mixture in 16 wells. Calculate the Z’ factor using the equation below; values greater than 0.7 are acceptable.
Note
Caution: Contact BellBrook Labs Technical Service for assistance if the calculated Z’ factor is less than 0.7.
Determine the ADP HiLyte647 Tracer Concentration
The Transcreener® ADP2 TR-FRET Red Assay requires detection of ADP in the presence of excess ATP (assuming initial velocity enzyme reaction conditions) using an antibody with a finite selectivity for the diphosphate vs. the triphosphate. The concentration of ADP HiLyte647 tracer determines the total assay window and the ADP detection range; the amount needed primarily depends upon the ATP concentration in the enzyme reaction.
Figure 3. Linear relationship between [ATP] and [ADP Tracer]. The tracer concentration can be calculated using the equation: y = 1.9x + 7.8
4.2.1 Calculate the Tracer Amount
As shown in Figure 3, the relationship between ATP and ADP HiLyte647 Tracer concentrations is linear. (Though shown for 0.1–100 µM ATP, the relationship is valid to 1,000 µM ATP.) Therefore, the quantity of ADP HiLyte647 Tracer for enzyme reactions that use between 0.1 μM and 1,000 μM ATP can be determined using the equation y = mx + b, where x = [ATP] (µM) in the 10 μL enzyme reaction, y = [ADP HiLyte647 Tracer] (nM) in the 10 μL of 1X ADP Detection Mixture, m (slope) = 1.9, and b (y-intercept) = 7.8. We recommend a final reaction volume of 20 μL.
For example, if you are using 3 µM ATP in a 10 µL enzyme reaction, the optimal ADP HiLyte647 Tracer concentration in the 1X ADP Detection Mixture (assuming 10 µL of ADP Detection Mixture was added to each 10 µL enzyme reaction) would be (1.9 × 3) + 7.8 = 13.6 nM.
4.2.2 Optimize the Tracer Concentration
Using the [ADP HiLyte647 Tracer] calculated using the equation in Figure 3 will produce excellent results for most users. If it does not produce the results you require, simply optimize the tracer concentration in a stepwise fashion using the [ADP HiLyte647 Tracer] (X) from the line as a starting point. Try performing a standard curve (see Section 7.1) at 0.5 × [Y], [Y], and 1.5 × [Y] tracer concentrations to find an assay window that suits your needs. See Section 6 for troubleshooting suggestions.
Optimize the Enzyme Concentration
Perform an enzyme titration to identify the optimal enzyme concentration for the Transcreener® ADP2 TR-FRET Red Assay. Use enzyme buffer conditions, substrate, and ATP concentrations that are optimal for your target enzyme and ADP HiLyte647 Tracer concentration calculated as described in Section 4.2. If a compound screen is planned, you should include the library solvent at its final assay concentration. We routinely use enzyme buffer containing 35 mM HEPES (pH 7.5), 4 mM MgCl2, 2 mM EGTA, 1% DMSO (test compound solvent), 0.015% Brij-35, and ATP. Run your enzymatic reaction at its requisite temperature and time period. Refer to Section 7.2 for the tolerance of different components for your buffer conditions.
4.3.1 Enzyme Titration Steps
To achieve the most robust assay and a high signal, the quantity of enzyme required to produce a 50–80% change in FRET signal is ideal (EC50 to EC80) for screening of large compound libraries and generating inhibitor dose-response curves (see Figure 4). To determine the EC80 enzyme concentration, use the following equation:
EC80 = (80 ÷ (100 – 80) )(1 ÷ hillslope) × EC50
Figure 4. Enzyme titration curve. The ideal range of enzyme concentrations is shown in red.
4.3.2 Enzyme Assay Controls
The enzyme reaction controls define the limits of the enzyme assay.
| Component | Notes | |
| 0% ATP Conversion Control | This control consists of the ADP Detection Mixture, the enzyme reaction components (without enzyme), and 100% ATP (0% ADP). It defines the upper limit of the assay window. | |
| 100% ATP Conversion Control | This control consists of the ADP Detection Mixture, the enzyme reaction components (without enzyme), and 100% ADP (0% ATP). It defines the lower limit of the assay window. | |
| Minus-Nucleotide Control and Minus-Substrate Control | To verify that the enzyme does not interfere with the detection module, perform an enzyme titration in the absence of nucleotide (i.e., ATP) or acceptor substrate. | |
| ADP/ATP Standard Curve | Although optional, an ADP/ATP standard curve can be useful to ensure day-to-day reproducibility and that the assay conditions were performed using initial rates. It can also be used to calculate product formed and inhibitor IC50 values. See Section 7.1 for a description of how to run the standard curve. | |
| Background Control | Use only 0.5X enzyme reaction conditions and Stop & Detect Buffer C. |
Run an Assay
1. Add the enzyme reaction mixture to test compounds and mix on a plate shaker.
2. Start the reaction by adding ATP and acceptor substrate, then mix. The final volume of the enzyme reaction mixture should be 10 µL. Incubate at a temperature and time ideal for the enzyme target before adding the ADP Detection Mixture.
3. Prepare 1X ADP Detection Mixture as follows:
ATP Concentration: Examples
| Component | 1 µM | 10 µM | 100 µM | Your Examples | |
| ADP2 Antibody-Tb | 100 µL | 100 µL | 100 µL | ||
| ADP HiLyte647 Tracer | 9.7 µL | 26.8 µL | 197.8 µL | ||
| 10X Stop & Detect Buffer C | 1,000 µL | 1,000 µL | 1,000 µL | ||
| Water | 8,890.3 µL | 8,873.2 µL | 8,702.2 µL | ||
| Total | 10,000 µL | 10,000 µL | 10,000 µL |
Final concentrations in the detection mixture should be 8 nM ADP2 Antibody-Tb, 1X Stop & Detect Buffer C, and the tracer concentration calculated using the equation in Figure 3. An example is shown below:
y=1.9x+7.8
| A | B | C | D | |
| ATP | 1 µM | 10 µM | 100 µM | |
| ADP HiLyte647 Tracer | 9.7 nM | 26.8 nM | 197.8 nM |
4. Add 10 µL of 1X ADP Detection Mixture to 10 µL of the enzyme reaction. Mix using a plate shaker.
5. Incubate at room temperature (20–25°C) for 1 hour and measure TR-FRET
General Considerations
Assay Types
5.1.1 Endpoint Assay
The Transcreener® ADP2 TR-FRET Red Assay is designed for endpoint readout. The Stop & Detect Buffer C contains EDTA to stop Mg2+-dependent enzyme reactions by chelating available Mg2+. Contact BellBrook Labs regarding stop buffers for non-Mg2+-dependent enzymes.
5.1.2 Real-Time Assay
You can perform real-time experiments by adding the ADP Detection Mixture, without the Stop & Detect Buffer C, directly to an enzyme reaction at initiation of the reaction. ADP detection equilibration time is not instantaneous, making it difficult to quantify ADP production; however, this method can provide insight into optimal enzyme concentration and incubation time. If Mn2+ or heavy metal ions, such as Cr3+, Co2+, Fe2+/3+, or Cu2+ are present, they can negatively quench the terbium chelate at high enough concentrations, so this method may not be possible for all enzymes. As an alternative, the Transcreener® ADP2 FP Assay is recommended to perform real-time assays. Note that the optimal ADP HiLyte647 Tracer concentration may change when EDTA is omitted from the reaction.
Reagent and Signal Stability
The Transcreener® technology provides a robust and stable assay method to detect ADP.
5.2.1 Signal Stability
The stability of the TR-FRET ratio assay window at 10% substrate conversion was determined after the addition of the ADP Detection Mixture to the standard samples. The ratio assay window at 10% substrate conversion (10 µM) remained constant (<10% change) for at least 24 hours at room temperature (20–25°C). If you plan to read TR-FRET on the following day, seal the plates to prevent evaporation.
5.2.2 ADP Detection Mixture Stability
The ADP Detection Mixture is stable for at least 8 hours at room temperature (20–25°C) before addition to the enzyme reaction (i.e., when stored on the liquid handling deck).
Troubleshooting
| Problem | Possible Causes and Solutions | |
| Low selectivity | Suboptimal tracer concentration
| |
| No change in TR-FRET observed | Low antibody/tracer activity
| |
| High background signal | Nonproductive ATP hydrolysis
|
Appendix
ADP/ATP Standard Curve
The standard curve mimics an enzyme reaction (as ATP concentration decreases, ADP concentration increases); the adenine nucleotide concentration remains constant. The ADP/ATP standard curve allows calculation of the concentration of ADP produced in the enzyme reaction and, therefore, the % ATP consumed (% ATP conversion). In this example, a 16-point standard curve was prepared using concentrations of ADP and ATP corresponding to 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 2.0, 2.5, 5.0, 10.0,25.0, 50.0, and 100% ATP conversion (see Table 1). Commonly, 8- to 12-point standard curves are used.
| % Conv. | ATP (µM) | ADP (µM) | |
| 100 | 0 | 100 | |
| 50 | 50 | 50 | |
| 25 | 75 | 25 | |
| 10 | 90 | 10 | |
| 5 | 95 | 5 | |
| 2.5 | 97.5 | 2.5 | |
| 1 | 99 | 1 | |
| 0.8 | 99.2 | 0.8 | |
| 0.6 | 99.4 | 0.6 | |
| 0.5 | 99.5 | 0.5 | |
| 0.4 | 99.6 | 0.4 | |
| 0.3 | 99.7 | 0.3 | |
| 0.2 | 99.8 | 0.2 | |
| 0.1 | 99.9 | 0.1 | |
| 0 | 100 | 0 |
Table 1. Concentrations of ATP/ADP to prepare a 16-point standard curve.
Figure 5. ATP/ADP standard curves.
A) Sample data for 0.1 μM, 1 μM, 10 μM, 100 μM, and 1,000 μM ADP/ATP standard curves. The nucleotide concentration reflects the amount in the enzyme reaction, prior to the addition of the ADP Detection Mixture. Curves are obtained in a final 20 μL assay volume consisting of 35 mM HEPES (pH 7.5), 2 mM MgCl2, 1 mM EGTA, 0.5% DMSO, 0.015% Brij-35, 10 mM EDTA, 4 nM ADP2 Antibody-Tb, ADP/ATP standards, and ADP HiLyte647 Tracer (concentration from equation in Figure 3) (n = 24). The data are plotted as FRET ratio and change in ratio vs. log [ADP] using 4-parameter nonlinear regression curve fitting. Alternatively, a 2-phase exponential decay and nonlinear regression can be used to present the data (GraphPad Prism).
B) Excellent Z’ values are obtained at <10% ATP conversion for the range of ATP concentrations used. Shown are 0.1 μM, 10 μM, and 1,000 μM ATP standard curves.
Use the following equations to calculate the Z’ factor:
Summary of Additive Effects on the Transcreener® ADP2 TR-FRET Assay
The assay window at 10% substrate conversion (10 µM ATP) remains constant (<10% change) when up to 10% DMSO, DMF, ethanol, acetonitrile, ethanol, or methanol are used in the enzyme reaction. Contact BellBrook Labs Technical Service for further reagent compatibility information.
Not all combination of these components have been tested together. Results may vary depending on your assay conditions.
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