Nov 10, 2025

Public workspaceTranscreener® dAMP FP Assay Technical Manual

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvw44bwlmk/v1
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvw44bwlmk/v1
Protocol Citation: info 2025. Transcreener® dAMP FP Assay Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw44bwlmk/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 23, 2025
Last Modified: November 10, 2025
Protocol Integer ID: 230614
Keywords: transcreener assay, human enzyme trex1, exonuclease enzymes such as trex1, degrading cytosolic dna, high throughput screening, exonuclease enzyme, assay, cytosolic dna, prime repair exonuclease, dna, trex1, sources of dna, deoxyadenylic acid
Abstract
The Transcreener® dAMP FP Assay is a far-red, competitive fluorescence polarization (FP) assay (Figure 1). The assay is designed to be used with exonuclease enzymes such as TREX1 (also known as Three Prime Repair Exonuclease 1), that produce the product dAMP (deoxyadenosine monophosphate, deoxyadenylic acid, or deoxyadenylate) by cleaving sources of DNA. An example is the human enzyme TREX1 which is the major exonuclease responsible for degrading cytosolic DNA.
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.
Troubleshooting
Introduction
The Transcreener® dAMP FP Assay is a far-red, competitive fluorescence polarization (FP) assay (Figure 1). The assay is designed to be used with exonuclease enzymes such as TREX1 (also known as Three Prime Repair Exonuclease 1), that produce the product dAMP (deoxyadenosine monophosphate, deoxyadenylic acid, or deoxyadenylate) by cleaving sources of DNA. An example is the human enzyme TREX1 which is the major exonuclease responsible for degrading cytosolic DNA.
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 Transcreener dAMP FP Assay provides the following benefits:
  • A simple single addition exonuclease activity assay capable of HTS.
  • Excellent data quality (Z’ ≥ 0.7) and signal (≥85 mP polarization shift) at dAMP ranges between 0.1-10 μM.
  • Sensitive detection of dAMP down to 10 nM and as high as 100 μM.
  • Far-red tracer further minimizes interference from fluorescent compounds and light scattering.

Figure 1. Schematic overview of the Transcreener dAMP FP Assay. The Transcreener dAMP Detection Mixture contains an dAMP AlexaFluor® 633 tracer bound to an dAMP antibody. dAMP produced by an exonuclease enzyme displaces the tracer, which rotates freely, causing a decrease in FP.

Product Specifications

ProductQuantityPart #
Transcreener® dAMP FP Assay1,000 assays*3028-1K
10,000 assays*3028-10K
*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 –20°C upon receipt.
Please recommend avoiding freeze thaw cycles for the best result. The assay has exhibited little or no signal change with up to 5 freeze thaw cycles. Please aliquot and store if not using multiple reagents at one time.
Use the reagents provided in this kit within 1 year from date of receipt.
Materials Provided

ComponentCompositionNotes
dAMP Antibody3.45 mg/mL solution in PBS with 5% glycerol*Mouse polyclonal antibody. Sufficient antibody is included in the kit to complete 1,000 assays (Part # 3028-1K) or 10,000 assays (Part # 3028-10K).
dAMP Alexa Fluor 633 Tracer400 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35Sufficient tracer is included in the kit to complete 1,000 assays (Part # 3028-1K) or 10,000 assays (Part # 3028-10K).
Stop & Detect Buffer B, 10X200 mM HEPES (pH 7.5), 400 m EDTA, and 0.2% Brij-35The Stop & Detect Buffer B 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 B at the time of FP measurement is 0.5X.
dAMP5 mM in H2O (pH 7.0)The dAMP in this kit can be used to generate a standard curve which can be used to convert mP values to dAMP 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
  • 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 dAMP assays are designed for use with purified enzyme preparations. Contaminating enzymes, can produce background signal and reduce the assay window.
  • Enzyme Buffer Components—User-supplied enzyme buffer components include enzyme, buffer, and test compounds.
  • Plate Reader—A multidetection microplate reader configured to measure FP of the dAMP Alexa Fluor 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, Safire 2™, and M1000.
  • 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.
  • Liquid Handling Devices—Use liquid handling devices that can accurately dispense a minimum volume of 0.5 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 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 dAMP FP Assay protocol consists of 3 steps (Figure 2). The protocol was developed for a 384-well format, using a 10 µL enzyme reaction and 20 µL complete assay volume when the plates are read. The use of different densities or reaction volumes will require changes in reagent quantities (see Section 7.3 for example reaction volumes). Once the instrument parameters and enzyme optimization are complete, the assay itself consists of a single step, simply add detection reagents to your enzyme reaction and read the plate.

Figure 2. An outline of the procedure. The procedure consists of 3 main steps with a mix-and-read assay format.

Set Up the Instrument
Becoming familiar with ideal instrument settings for FP is essential to the success of the Transcreener dAMP 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 dAMP 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 dAMP Alexa Fluor 633 Tracer at 1 nM in your enzyme buffer in a 20 µL complete assay. This mimics the 2-fold dilution when adding an equal volume of detection mixture to an enzyme reaction. As an example, the 1X dAMP Detection Mixture may contain 2 nM tracer. After adding this to the enzyme reaction, the concentration in the 0.5X, 20 µL complete assay would be 1 nM.

High FP Mixture Prepare the following solution.
ComponentStock ConcentrationComplete Assay ConcentrationExample: 25 AssaysYour Numbers
dAMP Antibody3.45 mg/mL*10.0 µg/mL1.45 µL**
dAMP Alexa Flour 633 Tracer400 nM1 nM1.25 µL**
Enzyme Buffer495.9 µL
Total500.0 µL
*Please note dAMP 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.
ComponentStock ConcentrationComplete Assay ConcentrationExample: 25 AssaysYour Numbers
dAMP Alexa Fluor 633 Tracer400 nM1 nM1.25 µL
Enzyme Buffer498.75 µL
Total500.0 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 dAMP 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 dAMP concentration produced in the enzyme reactions. We have determined optimal dAMP Antibody concentrations for up to 100 μM dAMP.
Using the dAMP Antibody concentration calculated in the chart below will produce excellent results for most users. If it does not produce the results you require a dAMP antibody titration might be required. Please refer to Section 7.2 for instructions on preparing the dAMP Antibody titration in the buffer system ideal for you enzyme target.

dAMP Concentration in 10 µL Enzyme Reaction dAMP Antibody Concentration in 10 µL 1X dAMP Detection Mixture
0.2 - 2 µM10.0 µg/mL
2 - 10 µM20.0 µg/mL
>10 µM60.0 µg/mL

Optimize the Enzyme Concentration
Perform an enzyme titration to identify the optimal enzyme concentration for the Transcreener dAMP 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. We have used an enzyme buffer containing 10 mM TRIS (pH 7.5), 7.5 mM MgCl2, 0.01% Brij-35, and 1% DMSO (test compound solvent). Run your enzymatic reaction at its requisite temperature and time period.
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 FP signal is ideal (EC50 to EC80) for screening of large compound libraries and generating inhibitor dose-response curves (see Figure 3). It is recommended to have at least a 100 mP shift to achieve a good assay window. Typically, an EC80 has been used with enzymes. To determine the EC80 enzyme concentration based on the EC50, use the following equation:

ECX = (X ÷ (100 – X) )(1 ÷ |hillslope| ) × EC50

Figure 3. Enzyme titration curve. Example enzyme titration with TREX1. 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.

ComponentNotes
No Inhibitor ControlThis control consists of the dAMP Detection Mixture, the enzyme reaction components with enzyme, and no inhibitor. This control will provide the dAMP formed from the reaction when screening.
No Enzyme ControlThis control consists of the dAMP Detection Mixture, the enzyme reaction components without enzyme. It defines the upper limit of the assay window.
Minus-DNA ControlTo verify that the enzyme does not interfere with the detection module, perform an enzyme titration in the absence of DNA.
dAMP Standard CurveAlthough optional, a standard curve can be useful to ensure day-to-day reproducibility. 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 ControlUse only 0.5X enzyme reaction conditions .



Run an Assay

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.

4.4.1 Experimental Samples 1. Add the enzyme to the test compounds at the desired concentration. The total volume of this mixture is 5 µL. Mix on a plate shaker. Incubate the enzyme inhibitor mixture for the desired time (typically at least 30 minutes). 2. Start the enzyme reaction by adding 5 µL of substrate, then mix. Concentrations may vary based on your experiment. Note: The final volume of the enzyme reaction mixture should be 10 µL for 384 well plates. Use 2X substrate, in 5 µL to achieve the appropriate final concentration. See Section 7.3 for a list of other plate formats. 3. It is recommended to incubate the enzyme reaction for 1 hour at room temperature. Please incubate at a temperature and time ideal for your experiment. 4. Prepare 1X dAMP Detection Mixture as follows:


1X dAMP Detection Mixture
ComponentStockDetection Mix Conc.Example Volume 2-10 µM dAMPYour Numbers
dAMP Antibody3.45 mg/mL20.0 µg/mL58 µL
dAMP Alexa Fluor 633 Tracer400 nM2 nM50 µL
10X Stop & Detect Buffer B10X1X1,000 µL
Water-- 8,892 µL
Total10,000 µL
5. Add 10 µL of 1X dAMP Detection Mixture to 10 µL of the enzyme reaction. Mix using a plate shaker. Note: After detection mixture is added to enzyme reaction the final concentration of components in a 20 µL complete assay will be 0.5X the dAMP Detection Mixture (0.5X Stop & Detect Buffer B, 1 nM tracer, and 10 µg/mL dAMP Antibody ). 6. Incubate at room temperature (20–25°C) for 90 minutes and measure FP.

4.4.2 dAMP Detection Controls These controls are used to calibrate the FP plate reader and are added to wells that do not contain enzyme.

ComponentNotes
Minus Antibody (Free Tracer) ControlThis control contains the dAMP Tracer without the dAMP Antibody and is set to low mP, typically between 20-50 mP depending on the instrument.
Minus Tracer ControlThis control contains the dAMP Antibody without the dAMP Tracer and is used as a sample blank for all wells. It contains the same dAMP Antibody concentration in all wells.

General Considerations

Assay Types
5.1.1. Endpoint Assay The Transcreener dAMP Assay Kit is designed for endpoint readout. The Stop & Detect Buffer B contains EDTA to stop Mg2+ dependent enzyme reactions by chelating available Mg2+.
5.1.2 Real-Time Assay Real-time experiments can be performed by substituting the Stop & Detect Buffer B, 10X (provided) with a detection buffer that does not contain EDTA. However, the equilibration time for the tracer, dAMP Antibody, and dAMP is at 80% after 15 minutes and stabilizes after 90 minutes. Real-time assay can be achieved but with precaution when converting mP to dAMP formation. Running a standard curve side by side with your real-time assay is a way to achieve accurate conversion. We recommend reading the plate after 90 minutes for screening and potency profiling for best results.
Reagent and Signal Stability
Transcreener technology provides a robust and stable assay method to detect dAMP.
5.2.1 Signal Stability The mP value at 5 µM dAMP 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 dAMP Detection Mixture Stability The dAMP Detection Mixture is stable for up to 8 hours at room temperature (20–25°C) before addition to the enzyme reaction (i.e., when stored on the liquid handling deck).
Frequently Asked Questions

QuestionPossible Solutions
No change in FP observedLow antibody/tracer activity or Δ mP signal.
  • The tracer and antibody are stable for up to 5 freeze-thaw cycles. For frequent use aliquot the antibody and tracer and store the aliquots at -20°C. Use a minimum of 10 μL aliquots.
  • Other components of the detection mix should not have multiple freeze-thaw cycles. Aliquot reagents and store at -20°C for future use.
Is a standard curve required every time I run an enzyme reaction?No, it is not required to run a standard curve. We recommend running the dAMP standard curve, if you want to convert the raw mP values to product formed (dAMP) or if you are determining Km or Vmax. 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 ATPases and nucleases in the lysates prohibits the use of Transcreener assays with lysates.
Why is my window with the enzyme very small?Example reasons for a diminished window
  • Be sure that the protein has activity. We recommend using enzyme from a commercial source as a positive control as needed.
  • Since the equilibration time of the assay is 90 minutes, start reading the plate at 90 min. Earlier time points may lead to diminished signal.

Appendix

dAMP Standard Curve
The standard curve mimics an enzyme reaction in which dAMP is formed. The standard curve allows calculation of the concentration of dAMP produced in the enzyme reaction. In this example, a 16-point standard curve was prepared using the concentrations of dAMP shown in Table 1. Commonly, 12- to 16-point standard curves are used.

Data PointdAMP (µM)
1200.000
266.667
322.222
47.407
52.469
60.823
70.274
80.091
90.030
100.010
110.0034
120.0011
130.00038
140.00013
150.00004
160.000


Figure 4. dAMP Standard Curve. Standard curve with varying dAMP Antibody concentrations to obtain ideal sensitivity.
Standard curves illustrate tuning the assay to the appropriate levels for the sensitivity desired. In this example 200 µM dAMP standard curves were completed using 5, 10, and 30 µg/mL final concentrations of dAMP antibody. Using the correct dAMP antibody for the amount of dAMP produced in the assay is imperative to achieving quality results. Use the following equations to calculate the Z’ factor:



Optimizing the dAMP 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 dAMP Antibody titration in the buffer system ideal for your enzyme target. This titration will determine the optimal antibody concentration for your assay conditions. The nucleotide substrate concentration in the enzyme reaction generally determines the appropriate concentration of dAMP Antibody. We recommend using the EC70 for 0.2 - 2 µM, EC85 for 2 - 20 µM, and EC95 for >10 µM range of dAMP produced by the enzyme for the selected dAMP concentration of antibody
7.2.1. Titrate the dAMP Antibody 1. Prepare the reaction buffer. Example: 10 mM TRIS, 7.5 mM MgCl2, and 0.01% Brij-35. Include desired substrate but omit the enzyme. 2. Dispense 10 µL of the reaction buffer into each well of columns 2–16. 3. Dispense 20 µL of dAMP Antibody (at 2 mg/mL concentration in the same reaction buffer) into each well of column 1. 4. Remove 10 μL from each well of column 1 and add it to the corresponding well of column 2. 5. Repeat step 4 for the remaining columns, thereby performing a 2-fold serial dilution across the plate to column 16. 6. Add 10 µL of 2 nM dAMP Alexa Fluor® 633 Tracer in 1X Stop & Detect Buffer B to each well. 7. Mix the plate, equilibrate at room temperature for 1.5 hours, and measure FP.

7.2.2 Calculate the Optimal dAMP Antibody Concentration To determine the dAMP antibody concentration for ECx, input the EC50 and hillslope values from a sigmoidal dose-response curve fit into the equation below. The dAMP Antibody is added to the 1X dAMP Detection Mixture at a concentration equivalent to 2 x [ECx].

ECX = (X ÷ (100 – X) )(1 ÷ |hillslope|) × EC50

Using the Assay with Different Volumes and Plate Formats

ComponentTotal VolumeEnzyme Reaction VolumedAMP Detection Mix Volume
96 Well Low Volume Plate50 µL25 µL25 µL
384 Well Low Volume Plate20 µL10 µL10 µL
1536 Well Low Volume Plate8 µL4 µL4 µL
Please check the working plate volumes from the manufacturer to ensure they are within the suggest volumes ranges of your plate.

Summary of Additive Effects on the Transcreener dAMP FP Assay
The assay window was determined to have limited effect with certain components when used under the recommended conditions. To determine the additive affects of a buffer component please test by titrating the component in the known concentration range. Use only the detection mix and a standard curve to determine the effect on assay performance. The maximum tolerance is defined as the mP reading at 0 µM and 10 µM dAMP where there was <3 standard deviation change observed at the listed concentration below. Signal was measured at 1.5 hours and 24 hours after dAMP detection mix was added.



a. mP at 0 µM or 10 µM dAMP where the mP signal increased or decreased <3 standard deviations of the plate controls at the listed concentration.
Not all combination of these components have been tested together. Results may vary depending on your assay conditions

Contact Information
Email: [email protected] Phone: 608.443.2400 Toll-Free: 866.313.7881 FAX: 608.441.2967