Nov 10, 2025
Enzolution™ TREX1 Assay System Technical Manual
- info 1
- 1BellBrook Labs LLC
- BellBrook LabsTech. support phone: +1 (608) 443-2400 email: [email protected]
Protocol Citation: info 2025. Enzolution™ TREX1 Assay System Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zz8dgqe/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: 230624
Keywords: enzymatic activity for trex1, purified human trex1, trex1 inhibitor, trex1 concentration, adjustment of the trex1 concentration, human trex1, degrading cytosolic dna, trex1, fp assay kit, cytosolic dna, dna substrate, stranded dna, stimulatory dna, preventing aberrant autoimmune response, ifn response in mammalian cell, assay, inhibitor dose response measurement, 45bp dsdna oligomer, interferon stimulatory dna, sting pathway, prime repair exonuclease, enzymatic activity, deoxynucleoside monophosphate, insensitive to dna, dna, releasing deoxynucleoside monophosphate, high throughput screening, 45bp dsdna oligomer from the listeria
Abstract
The Enzolution™ TREX1 Assay System is intended for use with the Transcreener® dAMP FP Assay Kit (Part #3028) to measure enzymatic activity for TREX1 (also known as Three Prime Repair Exonuclease 1). TREX1 is the major exonuclease responsible for degrading cytosolic DNA, and it plays an important role in preventing aberrant autoimmune responses in normal cells by acting as a checkpoint for the cGAS/STING pathway. It is a 3’-5’ exonuclease that cleaves single bases from the 5’ termini of double stranded or single stranded DNA, releasing deoxynucleoside monophosphates, including deoxy-AMP (dAMP). The Transcreener® dAMP FP assay enables sensitive detection of dAMP in a single addition, mix-and-read format that has been optimized and extensively validated for high throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers.
The Enzolution™ TREX1 Assay System provides all reagents required to screen and profile TREX1 inhibitors when used with the Transcreener® dAMP FP Assay Kit, including purified human TREX1 (amino acids 1-286, C-terminal 6xHis) and DNA Substrate. TREX1 is relatively non-specific for its DNA Substrate; the Enzolution™ TREX1 Assay System uses interferon stimulatory DNA (ISD), a 45bp dsDNA oligomer from the Listeria monocytogenes genome that induces a strong type I IFN response in mammalian cells. Note that we have optimized the assay using a saturating concentration of ISD, which makes the assay relatively insensitive to DNA-competitive inhibitors; use of lower ISD concentrations will require adjustment of the TREX1 concentration accordingly. Also, the protocol is configured for 384-well plates; use of different multiwell plate formats will require some additional optimization.
Troubleshooting
Introduction
The Enzolution™ TREX1 Assay System is intended for use with the Transcreener® dAMP FP Assay Kit (Part #3028) to measure enzymatic activity for TREX1 (also known as Three Prime Repair Exonuclease 1). TREX1 is the major exonuclease responsible for degrading cytosolic DNA, and it plays an important role in preventing aberrant autoimmune responses in normal cells by acting as a checkpoint for the cGAS/STING pathway. It is a 3’-5’ exonuclease that cleaves single bases from the 5’ termini of double stranded or single stranded DNA, releasing deoxynucleoside monophosphates, including deoxy-AMP (dAMP). The Transcreener® dAMP FP assay enables sensitive detection of dAMP in a single addition, mix-and-read format that has been optimized and extensively validated for high throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers.
The Enzolution™ TREX1 Assay System provides all reagents required to screen and profile TREX1 inhibitors when used with the Transcreener® dAMP FP Assay Kit, including purified human TREX1 (amino acids 1-286, C-terminal 6xHis) and DNA Substrate. TREX1 is relatively non-specific for its DNA Substrate; the Enzolution™ TREX1 Assay System uses interferon stimulatory DNA (ISD), a 45bp dsDNA oligomer from the Listeria monocytogenes genome that induces a strong type I IFN response in mammalian cells. Note that we have optimized the assay using a saturating concentration of ISD, which makes the assay relatively insensitive to DNA-competitive inhibitors; use of lower ISD concentrations will require adjustment of the TREX1 concentration accordingly. Also, the protocol is configured for 384-well plates; use of different multiwell plate formats will require some additional optimization.
Key Applications:
- Screening for TREX1 inhibitors
- Generating dose response curves and IC50 values for TREX1 inhibitors
- Kinetic and mechanistic analyses
Product Specifications
| Product | Quantity | Part # | |
| Enzolution™ TREX1 Assay System | 1,000 assays* | 3029-1K | |
| 10,000 assays* | 3029-10K |
*The exact number of assays depends on the enzyme reaction conditions. The kits are designed for use with 384-well plates, using 10 µL Enzyme Reaction and 20 µL Complete Assay volumes.
Storage
TREX1 should be stored at -80°C; other reagents can be stored at –20°C. Though we have confirmed that TREX1 is stable up to 2 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 | |
| TREX1 Enzyme | 0.1 mg/mL (3.2 µM)* solution in 50 mM TRIS (pH 8.0), 500 mM NaCl with 20% glycerol | Amino acids 1-286, C-terminal 6xHis, 31.3 kDa. Sufficient enzyme is included in the kit to complete 1,000 assays (Part # 3029-1K) or 10,000 assays (Part # 3029-10K). | |
| Interferon Stimulatory DNA | 25 μM in H2O | Supplied as a 45-bp annealed duplex interferon stimulatory DNA (ISD) oligomer. | |
| TREX1 Assay Buffer, 10X | 100 mM TRIS (pH 7.5), 75 mM MgCl2, 0.05% BSA, and 0.1% Brij-35 | Use the TREX1 Assay Buffer in the Enzyme Reaction and for inhibitor incubation. Changes to the assay buffer could affect TREX1 activity and/or detection of dAMP. | |
| 384-Well Low Volume Black Assay Plates | Corning #4514 | Black polystyrene non binding assay plates in either a 3-pack (1,000+ Assays) or a 30-pack (10,000+ Assays). We strongly recommend the use of these plates as inconsistent results have been observed with other plate formats. |
*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 nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Use nuclease free water such as: Invitrogen Part # AM9930 | |
| 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, Safire2™, and M1000. | |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense a submicroliter volumes into 384-well plates. | |
| Laboratoy Incubator | An incubator model that is capable of maintaining temperature stability at 30°C is required. |
Transcreener® dAMP Exonuclease Assay - SOLD SEPARATELY
| Component | Composition | Notes | |
| dAMP Antibody | 3.5 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 Tracer | 400 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35 | Sufficient 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, 10X | 200 mM HEPES (pH 7.5), 400 m EDTA, and 0.2% Brij-35 | The EDTA in the Stop & Detect Buffer B quenches the Enzyme Reaction by chelating Mg2+. Therefore, the assay should work as an end-point assay as long as the EDTA is at least equimolar to the Mg2+. The final concentrations of Mg2+ and EDTA in the Complete Assay are 7.5 mM and 20 mM, respectively. | |
| dAMP | 5 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. |
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 Transcreener® dAMP Assay Technical Manual Section 4.1).
3. Please read and understand the Transcreener® dAMP Assay Technical Manual prior to using with this kit.
Protocol
The methods described below are for single-addition, endpoint detection in 384 well plates using 10 μL TREX1 reactions and 10 μL of detection/quench reagents (final volume 20 μL when the plates are read). TREX1 reactions are initiated by the addition of the ISD Substrate and quenched by the addition of EDTA in the dAMP Detection Mix (see Figure 2) (TREX1 is a Mg-dependent enzyme). The use of different plate densities or reaction volumes will require changes in reagent quantities (see Section 5.1 for example reaction volumes). Additionally, the methods were designed for initial velocity detection of dAMP formation by TREX1 over a range of 0.2 to 20 μM dAMP; if desired, the dynamic range can be changed by adjusting the concentration of dAMP Ab, as described in the Transcreener® dAMP FP Assay Technical Manual.
10 µL Enzyme Reaction Components
| Component | As Provided | Working Stock | Final Concentration in 10 µL | |
| TREX1 Assay Buffer, 10X | 10X | 1X, Nuclease Free Water | 1X (10 mM TRIS pH 7.5, 7.5 mM MgCl2, 0.005% BSA, and 0.01% Brij-35) | |
| TREX1 Enzyme | 0.1 mg/mL (3.2 µM) | 2X in 1X TREX1 Assay Buffer | 1X (0.1 nM - 1.2 nM) | |
| Interferon Stimulatory DNA | 25 µM | 0.70 µM in 1X TREX1 Assay Buffer | 0.35 µM |
Table 1. TREX1 Enzyme Reaction Components. Concentrations are provided for the standard protocol using 5 µL of TREX1 Enzyme Mix and 5 µL of ISD Substrate Mix for the Enzyme Reaction.
1X dAMP Detection Mix - Add 10 µL Per Well
| Component | As Provided | Detection Mix Concentration | Final Concentration in 20 µL Complete Assay | Example Amounts for 10 mL Stock | |
| dAMP Antibody* | 3.5 mg/mL | 20 µg/mL | 10 µg/mL | 57.1 µL | |
| dAMP Alexa Fluor® 633 Tracer | 400 nM | 2 nM | 1 nM | 50.0 µL | |
| Stop & Detect Buffer B, 10X | 10X | 1X | 0.5X | 1,000.0 µL | |
| Nuclease Free Water | - | - | - | 8,892.9 µL |
*The exact concentration may vary from batch to batch. Please refer to the Certificate of Analysis for an accurate concentration.
Table 2. 1X dAMP Detection Mix Components. Volumes provided in the table are based on preparation of a 10 mL solution; adjust these appropriately for the desired volumes, including 10% extra for pipetting dead volumes.
Figure 2. An Outline of the Procedure. The TREX1 Enzyme Reaction is initiated by the addition of ISD. After the Enzyme Reaction incubation is completed, dAMP detection reagents are added (Transcreener® dAMP Antibody and Tracer) along with EDTA to quench the TREX1 reaction.
Setting Up a Standard Curve
Use of a standard curve for conversion of mP values to amount of dAMP formed allows quantitative measurement of TREX1 activity and accurate IC50 determinations; it is not typically done for screening at single concentrations. Here we describe preparation of a standard curve using 3X serial dilution from 200 μM to 0.0034 μM dAMP, which encompasses the appropriate range for this application.
Note: These concentrations represent the dAMP levels in the 10 µL TREX1 Enzyme Reaction; they will be diluted 2-fold by addition of detection reagents. The reagent volumes indicated below are sufficient for running the standard curve in duplicate plus excess for pipetting dead volume.
4.1.1 Detailed Methods
1. Prepare 600 µL 1X TREX1 Assay Buffer: dilute 60 µL TREX1 Assay Buffer, 10X in 540 µL Ultrapure Nuclease Free Water.
2. Prepare 30 µL of 400 µM dAMP: dilute 2.4 µL 5 mM dAMP stock in 27.6 µL 1X TREX1 Assay Buffer.
3. Prepare 160 µL of ISD Substrate Mix: dilute 4.5 µL Interferon Stimulatory DNA in 155.5 µL 1X TREX1 Assay Buffer.
4. Prepare 300 µL 1X dAMP Detection Mix based on the concentrations provided in Table 2: 30 µL 10X Stop & Detect Buffer B, 1.5 µL dAMP Alexa Fluor® 633 Tracer and 20 µg/mL dAMP Antibody in Ultrapure Nuclease Free Water.
Figure 3. Performing a Serial Dilution. Example 3-fold serial dilution of dAMP to generate a standard curve.
5. Add 7.5 µL of the 400 μM dAMP to well 1 (including replicates).
6. Add 5 μL of 1X TREX1 Assay Buffer to wells 2-12, DO NOT add the 1X TREX1 Assay Buffer to well 1.
7. Transfer 2.5 μL from well 1 to well 2 and mix by pipetting, then transfer 2.5 μL from well 2 to well 3 and mix by pipetting; repeat this serial dilution process until well 11 has received dAMP. Well 12 is to be used as a bIank and should correspond to 0 μM of dAMP on the standard curve.
IMPORTANT: After mixing the last well in the dilution series, remove 2.5 μL from that well only and discard, so that all the wells contain 5 μL final volume.
8. Add 5 μL of the ISD Substrate Mix to every well (1-12) then add 10 μL of 1X dAMP Detection mix to every well (1-12).
9. 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.
Figure 4. dAMP Standard Curve. Standard curve using 1X dAMP Detection Mix as described in Step 4 and Table 2.
Determining the Optimal Enzyme Concentration
Using EC80 enzyme concentration shown in the TREX1 Enzyme Certificate of Analysis should provide a robust signal that is within the linear range for dAMP formation. However, for best results, we suggest performing an enzyme titration to identify the optimal enzyme concentration, especially when running the assay in a different buffer system or with a different substrate concentration. The enzyme titration should be performed in duplicate and this example uses a 2X serial diultion. If a compound screen is planned, you should include the solvent (e.g., DMSO) at its final assay concentration. The Transcreener® dAMP FP Assay Kit has been shown to be able to tolerate up to 5% DMSO.
Note: TREX1 is a labile protein that is easily denatured; rapid or prolonged mixing should be avoided to preserve enzymatic activity.
4.2.1 Detailed Methods
1. Prepare 600 µL 1X TREX1 Assay Buffer: dilute 60 µL TREX1 Assay Buffer, 10X in 540 µL Ultrapure Nuclease Free Water.
2. Prepare 160 µL of 20 nM TREX1 Enzyme: dilute 1 µL TREX1 Enzyme in 159 µL 1X TREX1 Assay Buffer with gentle mixing.
3. Prepare 160 µL of ISD Substrate Mix: dilute 4.5 µL Interferon Stimulatory DNA in 155.5 µL 1X TREX1 Assay Buffer.
4. Add 10 µL of the 20 nM TREX1 to well 1 (including replicates).
5. Add 5 μL of 1X TREX1 Assay Buffer to wells 2-12, DO NOT add the 1X TREX1 Assay Buffer to well 1.
6. Transfer 5 μL from well 1 to well 2 and mix by pipetting, then transfer 5 μL from well 2 to well 3 and mix by pipetting; repeat this serial dilution process until well 11 has received TREX1 Enzyme. Well 12 is to be used as a blank and should not include enzyme.
IMPORTANT: After mixing the last well (11) in the dilution series, remove 5 μL from that well only and discard, so that all the wells contain 5 μL final volume.
7. Start the enzyme reaction by adding 5 μL of the ISD Substrate Mix to every well (1-12). Gently mix for 40 to 60 seconds on a plate shaker. Incubate at 30°C for 60 minutes.
8. Prepare 300 µL 1X dAMP Detection Mix based on the concentrations provided in Table 2: 30 µL 10X Stop & Detect Buffer B, 1.5 µL dAMP Alexa Fluor® 633 Tracer and 20 µg/mL dAMP Antibody in Ultrapure Nuclease Free Water.
9. Add 10 µL of 1X dAMP Detection mix to every well (1-12), in replicate.
10. 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.
Note: The reagent volumes indicated above are sufficient for running the enzyme titration in duplicate plus excess for pipetting dead volume. Scaling of volumes can be performed if necessary.
For detection of inhibitors at single concentration or in dose response mode, we recommend selecting an enzyme concentration that produces an 50-80% change in FP signal (EC50 to EC80) (see Figure 5) 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 dAMP formation (see Figure 6). The EC50 is provided by common graphing programs; the EC80 enzyme concentration can be calculated from the EC50, as follows:
ECX = (X ÷ (100 – X) )(1 ÷ |hillslope| ) × EC50
Figure 5. Enzyme Titration Curve. Example TREX1 Enzyme titration. The ideal range of enzyme concentrations is between EC50 and EC80; the specific concentration may vary depending on the enzyme lot.
Figure 6. Enzyme Titration Curve converted to dAMP formed. Raw polarization signal (mP) is converted to dAMP formed using a standard curve as described in Section 4.1. Only the linear portion of the graph is shown; interpolation was performed using GraphPad Prism.
Performing Single Compound Screening and Dose-Response Assays
4.3.1 Detailed Methods
1. Perform a serial dilution of test compounds with your method of choice. Add the TREX1 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: Final concentration should be based on the volume of the Enzyme Reaction.
2. Start the enzyme reaction by adding 5 µL of the ISD Substrate Mix. It is recommended to incubate the enzyme reaction at 30°C for 60 minutes.
Note: The final volume of the enzyme reaction mixture should be 10 µL for 384 well plates. See Section 5.1 for a list of other plate formats.
3. Following the 60 minute incubation, add 10 µL of 1X dAMP Detection Mix to the 10 µL Enzyme Reaction and mix the 20 µL Complete Assay using a plate shaker for 40 to 60 seconds.
4. Incubate at room temperature (20–25°C) for 90 minutes and measure FP
Figure 7. Dose-Response Curve. Example dose response curve with probe inhibitor Suramin.
Measuring Assay Robustness with Z’
By taking into account both dynamic range and data variability at the high and low ranges of the assay, the Z’ statistic provides a measure of the overall quality of an assay for HTS based on the usable signal or “assay window.” It is a dimensionless coefficient for the quality of the assay that is relevant for any assay, regardless of detection method or readout, without the intervention of test compounds. As a guideline a Z’ value of 0.5 or greater is generally considered to be indicative of a very good screening window for a biochemical assay. Z’ is determined using following formula, where complete reactions are untreated TREX1 enzymatic assays and No Enzyme are negative controls containing all components except TREX1; assays should be performed at n =16 for statistical significance.
Figure 8. Z’ Measurement. Complete assay is performed with and without TREX1 (n=16). Z’ is then calculated based on the formula from 4.4.1.
Appendix
Using the Assay with Different Volumes and Plate Formats
| Component | Total Volume | Enzyme Reaction Volume | dAMP Detection Mixture 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 |
Links to Applicable Application Notes
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