Nov 11, 2025
Transcreener® ADPR FP Assay Technical Manual
- info 1
- 1BellBrook Labs LLC
- BellBrook LabsTech. support phone: +1 (608) 443-2400 email: [email protected]
Protocol Citation: info 2025. Transcreener® ADPR FP Assay Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqppe3vk5/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 24, 2025
Last Modified: November 11, 2025
Protocol Integer ID: 230718
Keywords: sensitive detection of adpr, adpr from the hydrolysis, biochemical ht, adpr, adpr from the breakdown, high throughput screening, assay, glycohydrolase, adpr into amp, activity of human cd38, enzymatic reaction, coupling enzyme
Abstract
The Transcreener® ADPR FP Assay is a biochemical HTS assay for measuring the production of ADP-ribose (ADPR) in enzymatic reactions (Figure 1). The assay uses a Coupling Enzyme to convert ADPR into AMP, which is then detected using a far-red, competitive fluorescence polarization (FP) assay. As examples, the Transcreener® ADPR FP assay can be used to detect the activity of human CD38, which forms ADPR from the hydrolysis of NAD, or Poly (ADP-ribose) glycohydrolase (PARG), which produces ADPR from the breakdown of poly-ADP-ribose.
The Transcreener® ADPR FP assay is designed specifically for high throughput screening (HTS), with a single-addition, mix-and-read format. It is easy to integrate into automated HTS workflows, with outstanding reagent stability (deck stability > 16 hours, signal stability > 24 hours), sensitive detection of ADPR down to 10 nM and as high as 50 μM, and compatibility with commonly used multimode plate readers. Data quality is excellent (Z’ ≥ 0.7), and the assay uses a far-red tracer to minimize interference from fluorescent compounds and light scattering.
Troubleshooting
Introduction
The Transcreener® ADPR FP Assay is a biochemical HTS assay for measuring the production of ADP-ribose (ADPR) in enzymatic reactions (Figure 1). The assay uses a Coupling Enzyme to convert ADPR into AMP, which is then detected using a far-red, competitive fluorescence polarization (FP) assay. As examples, the Transcreener® ADPR FP assay can be used to detect the activity of human CD38, which forms ADPR from the hydrolysis of NAD, or Poly (ADP-ribose) glycohydrolase (PARG), which produces ADPR from the breakdown of poly-ADP-ribose.
The Transcreener® ADPR FP assay is designed specifically for high throughput screening (HTS), with a single-addition, mix-and-read format. It is easy to integrate into automated HTS workflows, with outstanding reagent stability (deck stability > 16 hours, signal stability > 24 hours), sensitive detection of ADPR down to 10 nM and as high as 50 μM, and compatibility with commonly used multimode plate readers. Data quality is excellent (Z’ ≥ 0.7), and the assay uses a far-red tracer to minimize interference from fluorescent compounds and light scattering.
Key Applications:
- Screening for enzyme inhibitors/activators
- Generating dose response curves and IC50 values for inhibitors.
- Kinetic and mechanistic analyses.
Figure 1. Schematic overview of the Transcreener ADPR FP Assay. ADPR produced by the target enzyme is converted to AMP by the Coupling Enzyme in real time. In the detection step, the Coupling Enzyme is quenched by EDTA and AMP displaces an Alexa Fluor® 633 tracer from the AMP2/GMP2 antibody, resulting in decreased fluorescence polarization.
Product Specifications
| Product | Quantity | Part # | |
| Transcreener® ADPR FP Assay | 1,000 assays* | 3030-1K | |
| 10,000 assays* | 3030-10K |
*The exact number of assays depends on the enzyme reaction conditions. The kits are designed for use with 384-well plates, using a 10 µL Enzyme Reaction and a 20 µL Complete Assay Volume.
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
ADPR-AMP Coupling Enzyme should be stored at -80°C; other reagents can be stored at –20°C. Though we have confirmed that the ADPR-AMP Coupling Enzyme is stable up to 3 freeze-thaw cycles, we recommend aliquoting the enzyme and snap-freezing for multiple uses to minimize loss of activity.
Use the reagents provided in this kit within 6 months from date of receipt.
Materials Provided
| Component | Composition | Notes | |
| AMP2/GMP2 Antibody | 1.26 mg/mL solution in PBS with 10% glycerol* | Sufficient antibody is included in the kit to complete 1,000 assays (Part # 3030-1K) or 10,000 assays (Part # 3030-10K). | |
| AMP2/GMP2 Alexa Fluor® 633 Tracer | 800 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35 | The final tracer concentration in the 20 µL Complete Assay is 4 nM. Sufficient tracer is included in the kit to complete 1,000 assays (Part # 3030-1K) or 10,000 assays (Part # 3030-10K). | |
| ADPR-AMP Coupling Enzyme | 400X ADPR-AMP Coupling Enzyme in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM DTT, 10% glycerol | Sufficient for 1,000 assays (Part # 3030-1K) or 10,000 assays (Part # 3030-10K) with Coupling Enzyme present in excess to ensure ADPR is completely converted to AMP. | |
| Stop & Detect Buffer B, 10X | 200mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35 | The Stop & Detect Buffer B components quench the Coupling Enzyme Reaction by chelating Mg2+. Therefore, it will work for any target enzyme, as long as the EDTA concentration is at least equimolar to the Mg2+. | |
| ADPR | 5 mM ADPR in deionized water, pH 7.0 | The ADPR in this kit can be used to create a standard curve to convert mP values to ADPR product formed. |
*The exact concentration may vary from batch to batch. Please refer to the Certificate of Analysis for an accurate concentration.
Materials Required but Not Provided
| Component | Notes | |
| Ultrapure Nuclease Free Water | Some deionized water systems are contaminated with enzymes that can degrade both nucleotide substrates and products, reducing assay performance. Use nuclease free water such as: Invitrogen Part # AM9930 | |
| Enzyme | The Transcreener® ADPR FP Assay is designed for use with purified enzymes capable of producing ADPR from a substrate. Contaminating enzymes, such as phosphatases or nucleotidases, can produce background signal and reduce the assay window. | |
| Substrate | The Transcreener® ADPR FP Assay is designed for use with purified enzymes capable of producing ADPR from a substrate. Substrate impurities and degradation may produce background signal and reduce the assay window. | |
| Plate Reader | A multimode microplate reader configured to measure FP of the AMP2/GMP2 AlexaFluor® 633 Tracer is required. Transcreener® FP Assays have been successfully used on the following instruments: BioTek Synergy™2 and Synergy™4; BMG Labtech PHERAstar® Plus and CLARIOstar® Plus; Molecular Devices SpectraMax™ Paradigm; Perkin Elmer EnVision® and ViewLux; and Tecan Infinite® F500, Safire2™, and M1000. | |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense submicroliter volumes into 384-well plates. | |
| Assay Plates | It is important to use assay plates that are entirely black with a nonbinding surface. We recommend Corning® 384-well plates (Cat. # 4514). The suggested plate has a square well top that enables easier robotic pipetting and a round bottom that allows good Z’ factors. It has a recommended working volume of 15–20 µL. |
Note
Note: Contact BellBrook Labs Technical Service for suppliers and catalog numbers for buffer components, and additional information regarding setup of FP instruments.
Before You Begin
1. Read the entire protocol and note any reagents or equipment needed (see Section 2.2).
2. Check the FP instrument and verify that it is compatible with the assay being performed (see Section 4.1).
Protocol
The Transcreener® ADPR FP Assay kit optimization follows a simple format. First, the instrument is configured with proper parameters to ensure a successful data readout (Section 4.1). This is followed by an optimization of the antibody concentration to obtain the desired dynamic range (Section 4.2). Next, an optimal enzyme concentration is determined to provide a good assay window while meeting initial velocity requirement (Section 4.3). Once the instrument parameters, antibody, and enzyme optimization are complete, the assay itself consists of a simple mix-and-read protocol (Section 4.4).
The assay protocol in Section 4.4 was developed for a 384-well format, using a 10 µL Enzyme Reaction and 20 µL Complete Assay volume when the plates are read (Figure 2). The 10 µL Enzyme Reaction is composed of two components: 5 µL of the target enzyme diluted to the desired concentration and 5 µL of a preparation containing both the Coupling Enzyme and the required substrate. The Coupling Enzyme working concentration is 1X in 10 µL Enzyme Reaction, which is present in at least 5x excess over what is required for complete conversion of ADPR to AMP in real time. Once the Enzyme Reaction is complete, simply add 10 µL of 1X AMP Detection Mix to your enzyme reaction and read the plate. The use of different densities or reaction volumes will require changes in reagent quantities (see Section 7.3 for example reaction volumes).
Note: Tracer concentration remains constant at 4 nM in the 20 μL Complete Assay regardless of changes to other reaction conditions. It is not recommended that this parameter be changed.
Figure 2. An outline of the Assay Protocol. The target enzyme reaction is run in the presence of Coupling Enzyme, so that ADPR is converted to AMP in real time. After the Enzyme Reaction incubation is complete, AMP detection reagents are added (Transcreener® AMP2/GMP2 Antibody and Tracer) along with EDTA to quench the Coupling Enzyme.
Set Up the Instrument
Becoming familiar with ideal instrument settings for FP is essential to the success of the Transcreener® ADPR FP Assay.
4.1.1 Verify That the Instrument Measures FP
Ensure that the instrument is capable of measuring FP (not simply fluorescence intensity) of the AMP2/GMP2 AlexaFluor 633 Tracer.
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 mP Window for the Instrument
Measuring high (tracer + antibody) and low (free tracer) FP will define the maximum assay window of your specific instrument. Prepare High and Low FP Mixtures in quantities sufficient to perform at least 6 replicates for each condition.
Use AMP2/GMP2 Alexa Fluor 633 Tracer at 4 nM in a 20 µL Complete Assay. This mimics the 2-fold dilution when adding an equal volume of 1X AMP Detection Mix to an Enzyme Reaction.
High FP Mixture
| Component | As Provided | Final Concentration in 20 µL Complete Assay | Example: 25 Assays | Your Numbers | |
| AMP2/GMP2 Antibody | 1.26 mg/mL* | 5 µg/mL | 2.4 µL** | ||
| AMP2/GMP2 Alexa Fluor 633 Tracer | 800 nM | 4 nM | 3 µL | ||
| Stop & Detect Buffer B | 10X | 0.5X | 30 µL | ||
| Water | 564.6 µL | ||||
| Total | 600.0 µL |
*Please note AMP2/GMP2 Antibody concentration varies by lot number. This is an example and should be adjusted based on stock concentration accordingly.
**Pipetting small sample volumes accurately requires the correct equipment and proper technique. An extra dilution step may be required to ensure accuracy.
Low FP Mixture
Prepare the following solution.
| Component | As Provided | Final Concentration in 20 µL Complete Assay | Example: 25 Assays | Your Numbers | |
| AMP2/GMP2 Alexa Fluor 633 Tracer | 800 nM | 4 nM | 3 µL | ||
| Stop & Detect Buffer B | 10X | 0.5X | 30 µL | ||
| Water | 567 µL | ||||
| Total | 600.0 µL |
4.1.3 Measure the FP
Subtract the Low FP Mixture readings from the corresponding High FP Mixture readings. The difference between the low and high FP values should be >100 mP.
Note
Caution: Contact BellBrook Labs Technical Service for assistance if the assay window is <100 mP.
Determine Optimal AMP2/GMP2 Antibody Concentration
The antibody is the only assay component that requires adjustment for different reaction conditions. Its concentration will define the dynamic range of the assay, and it should be adjusted based on the ADPR concentration produced in the Enzyme Reaction. We have determined optimal AMP2/GMP2 Antibody concentrations for up to 50 μM ADPR.
Using the AMP2/GMP2 Antibody concentration calculated in the chart below will produce excellent results for most users. If it does not produce satisfactory results, an AMP2/GMP2 antibody titration in your specific conditions is recommended. Please refer to Section 7.2 for instructions on preparing the AMP2/GMP2 Antibody titration in the buffer system ideal for your target enzyme.
| ADPR Concentration in 10 µL Enzyme Reaction | AMP2/GMP2 Antibody Concentration in 10 µL 1X AMP Detect Mix | |
| 0.2 - 0.7 µM | 2 µg/mL | |
| 0.7 - 3 µM | 10 µg/mL | |
| 3 - 10 µM | 50 µg/mL | |
| >10 µM | 100 µg/mL |
Optimize the Enzyme Concentration
Perform an enzyme titration to identify the optimal enzyme concentration for the Transcreener ADPR FP Assay. Use enzyme buffer conditions and substrate concentrations that are optimal for your enzyme and experimental goals. If a compound screen is planned, you should include the library solvent at its final assay concentration. Run your enzymatic reaction at its requisite temperature and time period.
4.3.1 Enzyme Titration Considerations
For detection of inhibitors at single concentration or in dose response mode, we recommend selecting an enzyme concentration that produces a 50–80% change in FP signal (EC50 to EC80) (see Figure 3) and an assay window of at least 100 mP. This will result in initial velocity conditions, which correspond to the linear phase of the reaction after conversion of mP values to ADPR formed. The EC50 is provided by common graphing programs; the EC80 enzyme concentration can be calculated from the EC50, as follows:
EC80 = (80 ÷ (100 – 80) )(1 ÷ |hillslope|) × EC50
Figure 3. Enzyme titration curve. Example enzyme titration with CD38. The ideal range of enzyme concentrations is between EC50 and EC80; the specific concentration may vary depending on the enzyme lot.
4.3.2 Enzyme Assay Controls
The enzyme reaction controls define the limits of the enzyme assay.
| Component | Notes | |
| No Inhibitor Control | This is a complete Enzyme Reaction with all detection components. It provides the maximum signal (minimal FP value) for an uninhibited enzyme reaction. | |
| No Enzyme Control | This contains all Enzyme Reaction components in the absence of the target enzyme. It provides the minimal signal (maximal FP value), mimicking 100% Inhibition. | |
| Minus-Substrate Control | This is an alternative to a No Enzyme control; it may be more appropriate for enzymes that produce some background signal that is not substrate-dependent. |
Performing Single Compound Screening and Dose-Response Assays
4.4.1 Experimental Samples
1. Perform a serial dilution of test compound with your method of choice. Add the enzyme to the test compounds at the desired concentration so that the total volume of this mixture is 5 µL. Mix gently on a plate shaker for 40 to 60 seconds. Preincubate the Enzyme Inhibitor Mix for the desired time (typically at least 30 minutes) at room temperature to allow equilibration of the E-I complex.
Note
Note: This is an example of running an assay for HTS or to obtain a dose response. Your volumes and concentrations may vary. It is important to have a 1:1 ratio of Enzyme Mix and Detection Mix for the final assay readout.
2. Start the enzyme reaction by adding 5 µL of the Substrate/Coupling Enzyme Mix then mix gently on a plate shaker for 40 to 60 seconds. It is recommended to incubate the enzyme reaction at 30°C for 60 minutes.
Note: Prepare the Substrate/Coupling Enzyme Mix right before use to avoid substrate degradation. Coupling Enzyme concentration is 2X in the Substrate/Coupling Enzyme Mix and 1X in the 10 μL Enzyme Reaction.
3. Prepare 1X AMP Detection Mix as follows:
1X AMP Detection Mix - Add 10 µL Per Well
| Component | As Provided | Detection Mix Concentration | Final Concentration in 20 µL Complete Assay | Final Volume in AMP Detect Mix | |
| AMP2/GMP2 Antibody | 1.26 mg/mL | 10 µg/mL | 5 µg/mL | 79.4 µL | |
| AMP2/GMP2 Alexa Fluor 633 Tracer | 800 nM | 8 nM | 4 nM | 100 µL | |
| Stop & Detect Buffer B | 10X | 1X | 0.5X | 1000 µL | |
| Water | 8,820.6 µL | ||||
| Total | 10,000 µL |
4. Following the 60 minute incubation, add 10 µL of 1X AMP Detection Mix and mix gently on a plate shaker for 40 to 60 seconds.
Note: After Detection Mix is added to Enzyme Reaction the final concentration of components in the 20 µL Complete Assay will be 0.5X the Detection Mix (4 nM tracer, 5 µg/mL AMP2/GMP2 Antibody, and 0.5X Stop & Detect Buffer B).
5. Incubate at room temperature (20–25°C) for 90 minutes to allow equilibration of assay components and measure FP.
4.4.2 AMP Detection Controls
These controls are used to calibrate the FP plate reader and are added to wells that do not contain
enzyme.
| Component | Notes | |
| Minus Antibody (Free Tracer) Control | This control contains 4 nM AMP2/GMP2 Tracer in the 20 μL Complete Assay without the AMP2/GMP2 Antibody and is set to low mP, typically between 20-50 mP depending on the instrument. | |
| Minus Tracer Control | This control contains the AMP2/GMP2 Antibody without the AMP2/GMP2 Tracer and is used as a sample blank for all wells. It contains the same AMP2/GMP2 Antibody concentration that will be used in the assay. |
General Considerations
Endpoint Assay
The Transcreener® ADPR FP Assay is designed for endpoint readout. The Stop & Detect Buffer B contains EDTA, which quenches the coupling enzyme by chelating Mg2+.
Reagent and Signal Stability
The Transcreener® technology provides a robust and stable assay method to detect ADPR.
5.2.1 Signal Stability
The stability of the FP assay window at 10% substrate conversion was determined after the addition of the 1X AMP Detection Mix to the standard samples. The FP assay window at 10% substrate conversion remained constant (<10% Change) for at least 24 hours at room temperature (20–25°C). If you plan to read FP on the following day, seal the plates to prevent evaporation.
5.2.2 AMP Detection Mix Stability
The AMP Detection Mix is stable for at least 16 hours at room temperature (20–25°C). If you prepare the AMP Detection Mix more than 30 minutes before addition, store it on ice or at 4°C until needed to help decrease the equilibration time.
Frequently Asked Questions
| Question | Possible Solutions | |
| No change in FP observed | Low antibody/tracer activity or Δ mP signal.
| |
| Is a standard curve required? | No, it is not required to run a standard curve. We recommend running the ADPR standard curve if you want to convert raw mP values to product formed. While designing a standard curve, make sure that most of the points are within the area of interest (initial velocity conditions). We do not recommend using a standard curve from previous experiments, rather generate a new curve with each experiment to achieve the most accurate result. | |
| Can this assay be used with cell lysates? | The assay will only work with purified recombinant protein. The presence of nucleases in the lysates prohibits the use of Transcreener assays with lysates. | |
| High background signal or change in signal after incubation with detection mixture. | Interference from impurities
|
Appendix
Standard Curve
The standard curve mimics any enzyme reaction where ADPR is formed and then converted to AMP. In this example a 16-point standard curve was prepared using the concentration of ADPR shown in Table 1. Commonly, 12-16 point standard curves are used. You may choose to not run a standard curve depending on your experiment.
| Data Point | ADPR (µM) | |
| 1 | 100.000 | |
| 2 | 50.000 | |
| 3 | 25.000 | |
| 4 | 12.500 | |
| 5 | 6.250 | |
| 6 | 3.125 | |
| 7 | 1.563 | |
| 8 | 0.781 | |
| 9 | 0.391 | |
| 10 | 0.195 | |
| 11 | 0.098 | |
| 12 | 0.049 | |
| 13 | 0.024 | |
| 14 | 0.012 | |
| 15 | 0.006 | |
| 16 | 0.003 |
Table 1. ADPR Standard Curve. Standard curve to help convert raw mP values to product formed.
Figure 4. ADPR Standard Curve. Standard curve with varying AMP2/GMP2 Antibody concentrations, in 10 μL 1X AMP Detection Mix, to obtain ideal sensitivity.
Standard curves illustrate tuning the assay to the appropriate levels for the sensitivity desired. In this example, 100 µM ADPR standard curves were completed using 2, 10, 50, and 100 µg/mL final concentrations of AMP2/GMP2 antibody in the 10 μL 1X AMP Detection Mix. Using the correct antibody concentration for the amount of ADPR produced in the assay is imperative to achieving quality results. Use the following equations to calculate the Z’ factor:
Optimizing the AMP2/GMP2 Antibody Concentration
Using an antibody concentration from the chart in Section 4.2 will produce excellent results for most users. If it does not produce the results you require, we recommend that you perform an AMP2/GMP2 Antibody titration in the buffer system ideal for your enzyme target. This titration will determine the optimal antibody concentration for your assay conditions. The substrate concentration in the enzyme reaction generally determines the appropriate concentration of AMP2/GMP2 Antibody. We recommend using a concentration of antibody equivalent to the EC70 to EC85 acquired from the titration.
7.2.1. Titrate the AMP2/GMP2 Antibody
1. Prepare the reaction buffer. Example: 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.001% BSA and 0.01% Brij-35. Include desired substrate but omit the enzyme.
2. Add 10 µL of the reaction buffer to wells 2–16 (including replicates). Do not add reaction buffer to well 1.
3. Add 20 µL of AMP2/GMP2 Antibody (at 0.2 mg/mL concentration in the same reaction buffer) to well 1 of each replicate.
4. Transfer 10 μL from well 1 to well 2 and mix by pipetting, then transfer 10 μL from well 2 to well 3 and mix by pipetting; repeat this serial dilution process until well 16 has received AMP2/GMP2 Antibody.
IMPORTANT: After mixing the last well in the dilution series, remove 10 μL from that well only and discard, so that all the wells contain 10 μL final volume.
5. Add 10 µL of 8 nM AMP2/GMP2 Alexa Fluor® 633 Tracer in 1X Stop & Detect Buffer B to each well.
6. Gently mix on a plate shaker for 40 to 60 seconds and then allow it to incubate at room temperature for 90 minutes before reading.
7.2.2 Calculate the Optimal AMP2/GMP2 Antibody Concentration
To determine the AMP2/GMP2 antibody concentration for ECx, input the EC50 and hillslope values from a sigmoidal dose-response curve fit into the equation below.
ECX = (X ÷ (100 – X) )(1 ÷ |hillslope|) × EC50
Using the Assay with Different Volumes and Plate Formats
| Component | Total Volume | Enzyme Reaction Volume | 1X AMP Detection Mix Volume | |
| 96 Well Low Volume Plate | 50 µL | 25 µL | 25 µL | |
| 384 Well Low Volume Plate | 20 µL | 10 µL | 10 µL | |
| 1536 Well Low Volume Plate | 8 µL | 4 µL | 4 µL |
Please check the working plate volumes from the manufacturer to ensure they are within the suggest volumes ranges of your plate.
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