Nov 17, 2025
Enzolution™ DDX5 ATPase 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™ DDX5 ATPase Assay System Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqp1b5vk5/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 31, 2025
Last Modified: November 17, 2025
Protocol Integer ID: 231266
Keywords: ddx5 atpase assay, ddx5 atpase, DDX5 ATPase assay, DDX5 ATPase assay system, dependent rna helicase activity of ddx5, atpase assay system, purified human ddx5, ddx5 inhibitor, transcreener adp2 assay, transcreener adp2 assay kit, rna helicase activity, dependent rna helicase activity, dead box helicase protein, formation by the rna helicase activity, assay, human ddx5, atp, ddx5, rna, conserved motif asp, inhibitor dose response measurement, dna duplex
Abstract
The Enzolution™ DDX5 Assay System is intended for use with the Transcreener® ADP2 Assay Kits to measure the ATP-dependent RNA helicase activity of DDX5. DDX5, also known as p68, is a DEAD Box helicase protein characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), that catalyzes ATP-dependent unwinding of RNA-RNA and RNA-DNA duplexes. ADP formation by the RNA helicase activity of DDX5 can be easily measured by the Transcreener ADP2 Assay, a far-red, competitive fluorescence assay that enables single addition, mix-and-read detection in a continuous or endpoint format. The assay has been optimized and extensively validated for high throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers.
The Enzolution™ DDX5 Assay System provides all reagents required to screen and profile DDX5 inhibitors, including the purified human DDX5 (aa 40-475) and yeast RNA, when used with the Transcreener ADP2 Assay Kits, which are available with FP, FI and TR-FRET readouts. The protocol is configured for 384-well plates; use of different multi-well plate formats will require adjustment of reagents concentrations utilized in the assay.
Troubleshooting
Introduction
The Enzolution™ DDX5 Assay System is intended for use with the Transcreener® ADP2 Assay Kits to measure the ATP-dependent RNA helicase activity of DDX5. DDX5, also known as p68, is a DEAD Box helicase protein characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), that catalyzes ATP-dependent unwinding of RNA-RNA and RNA-DNA duplexes. ADP formation by the RNA helicase activity of DDX5 can be easily measured by the Transcreener ADP2 Assay, a far-red, competitive fluorescence assay that enables single addition, mix-and-read detection in a continuous or endpoint format. The assay has been optimized and extensively validated for high throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers.
The Enzolution™ DDX5 Assay System provides all reagents required to screen and profile DDX5 inhibitors, including the purified human DDX5 (aa 40-475) and yeast RNA, when used with the Transcreener ADP2 Assay Kits, which are available with FP, FI and TR-FRET readouts. The protocol is configured for 384-well plates; use of different multi-well plate formats will require adjustment of reagents concentrations utilized in the assay.
Key Applications:
- Screening for DDX5 inhibitors (or activators)
- Generating dose response curves and IC50 values for DDX5 inhibitors
- Kinetic and mechanistic analyses
Figure 1. Schematic Overview of the Enzolution™ DDX5 Assay System with the Transcreener ADP2 Assays.
For FP readout (A): ADP produced by the RNA helicase activity of DDX5 displaces an Alexa Fluor® 633 Tracer from the ADP2 antibody, resulting in decreased fluorescence polarization.
For TR-FRET readout (B): ADP produced by the RNA helicase activity of DDX5 displaces a HiLyte647 Tracer from the ADP2 Antibody conjugated to terbium (Tb), resulting in a decrease in TR-FRET.
For FI readout (C): ADP produced by the RNA helicase activity of DDX5 displaces an Alexa Fluor® 594 Tracer from the ADP2 Antibody conjugated to an IRDye® QC-1 quencher, resulting in an increase in fluorescence intensity.
Product Specifications
| Product | Quantity | Part # | Part # | |
Enzolution™ DDX5 ATPase Assay System | 1,000 assays* | FP | 3039-1K-FP | |
| FI | 3039-1K-FI | |||
| TR-FRET | 3039-1K-TR | |||
| 10,000 assays* | FP | 3039-10K-FP | ||
| FI | 3039-10K-FI | |||
| TR-FRET | 3039-10K-TR |
*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.
Storage
DDX5 Enzyme and yeast RNA should be stored at -80°C; other reagents can be stored at –20°C. Though we have confirmed that the DDX5 Enzyme and the yeast RNA are stable and maintain greater than 80% activity up to 5 freeze-thaw cycles, we recommend aliquoting the reagents 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 | |
| DDX5 Enzyme | 0.1 mg/mL (1.91 µM)* in 50 mM Tris-HCl, 100 mM NaCl, 1 mM TCEP, 1 mM EDTA, 10% Glycerol, pH 7.5 | Truncated (amino acids 40-475), N-Terminal 6xHis, 52.43 kDa. Sufficient enzyme is included in the kit to complete at least 1,000 assays (Part # 3039-1K) or 10,000 assays (Part # 3039-10K). | |
| Yeast RNA | 10 mg/mL in H2O | Yeast RNA purified from Torulla. Storage solution is nuclease-free water. | |
| Enzyme Assay Buffer D, 10X | 500 mM TRIS (pH 7.5), 20 mM MgCl2, 0.1% Triton X-100 | Use Enzyme Assay Buffer D in the Enzyme Reaction and for preincubation with inhibitors. Changes to the assay buffer could affect enzyme activity and/or detection of ADP. | |
| 384-Well Low Volume Assay Plates | Corning #4514 -FP and FI Only Corning #4513 – TR-FRET Only | Polystyrene non-binding surface 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 plates. |
*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 multimode microplate reader configured to measure FP, FI, or TR-FRET is required. Transcreener Assays have been validated 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 sub-microliter volumes into 384-well plates. | |
| Laboratory Incubator | An incubator model that is capable of maintaining temperature stability at 30°C is required. |
Transcreener® ADP2 FP Assay - SOLD SEPARATELY
| Component | Composition | Notes | |
| ADP2 Antibody | 4 mg/mL solution in PBS with 10% glycerol* | 1,000 assays (Part # 3010-1K) or 10,000 assays (Part # 3010-10K). | |
| ADP2 Alexa Fluor® 633 Tracer | 400 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35 | 1,000 assays (Part # 3010-1K) or 10,000 assays (Part # 3010-10K). | |
| Stop & Detect Buffer B, 10X | 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35 | The EDTA in the Stop & Detect Buffer B quenches the DDX5 Enzyme Reaction by chelating Mg2+. The final concentrations of Mg2+ and EDTA in the Complete Assay are 2 mM and 20 mM, respectively. | |
| ATP | 5 mM | The ATP supplied in this kit can be used for the DDX5 Enzyme Reaction and the ATP/ADP standard curve. | |
| ADP | 5 mM | The ADP is used for the ATP/ADP standard curve. |
Transcreener® ADP2 FI Assay - SOLD SEPARATELY
| A | B | C | |
| ADP2 Antibody-IRDye® QC-1 | 1.4 mg/mL solution in 100 mM KH2PO4 (pH 8.5)* | 1,000 assays (Part # 3013-1K) or 10,000 assays (Part # 3013-10K). | |
| ADP2 Alexa Fluor® 594 Tracer | 800 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35 | 1,000 assays (Part # 3013-1K) or 10,000 assays (Part # 3013-10K). | |
| Stop & Detect Buffer B, 10X | 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35 | The EDTA in the Stop & Detect Buffer B quenches the DDX5 Enzyme Reaction by chelating Mg2+. The final concentrations of Mg2+ and EDTA in the Complete Assay are 2 mM and 20 mM, respectively. | |
| ATP | 5 mM | The ATP supplied in this kit can be used for the DDX5 Enzyme Reaction and the ATP/ADP standard curve. | |
| ADP | 5 mM | The ADP is used for the ATP/ADP standard curve. |
Transcreener® ADP2 TR-FRET Assay - SOLD SEPARATELY
| A | B | C | |
| ADP2 Antibody-Terbium Conjugate | 800 nM solution in HEPES-buffered saline | 1,000 assays (Part # 3011-1K) or 10,000 assays (Part # 3011-10K). | |
| ADP HiLyte647 Tracer | 10 µM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35 | 1,000 assays (Part # 3011-1K) or 10,000 assays (Part # 3011-10K). | |
| Stop & Detect Buffer C, 10X | 500 mM HEPES (pH 7.5), 200 mM EDTA, and 0.2% Brij-35 | The EDTA in the Stop & Detect Buffer C quenches the DDX5 Enzyme Reaction by chelating Mg2+. The final concentrations of Mg2+ and EDTA in the Complete Assay are 2 mM and 10 mM, respectively. | |
| ATP | 5 mM | The ATP supplied in this kit can be used for the DDX5 Enzyme Reaction and the ATP/ADP standard curve. | |
| ADP | 5 mM | The ADP is used for the ATP/ADP standard curve. |
*The exact concentration may vary from batch to batch. Please refer to the Certificate of Analysis for an accurate concentration.
Before You Begin
1. Read the entire protocol and note any reagents or equipment needed (see Section 2.2).
2. Check the plate reader and verify that it is compatible with the Transcreener® ADP2 Assays. Full list of compatible plate readers and settings.
3. Please read and understand the Transcreener ADP2 Assay Technical Manuals prior to use with this kit.
Protocol
The methods described below are for single-addition, endpoint detection: the DDX5 Enzyme Reaction is quenched by the addition of EDTA along with the detection reagents (see Figure 2). The methods were designed for 384-well plates using a 10 μL DDX5 Enzyme Reaction and 10 μL of detection/quench reagents (final volume 20 μL when the plates are read). The use of different plate densities or reaction volumes will require changes in reagent quantities (see Section 5.1 for example reaction volumes).
The methods were optimized for initial velocity detection of ADP formation (≤ 20% conversion of ATP to ADP) by DDX5 with a sub-Km concentration of ATP (100 μM) and a saturating concentration of yeast RNA (1 mg/mL). These conditions will ensure sensitive detection of inhibitors that compete with ATP while minimizing the effects of compounds that inhibit DDX5 via non-specific interactions with RNA. Significant changes in the ATP concentration may require optimization of the ADP2 Antibody concentration (in the case of the FP and FI readout modes) or the ADP Tracer (in the case of the TR-FRET readout mode) to adjust the dynamic range as described in the Transcreener ADP2 Assay Manual.
Note: Antibody (TR-FRET) and Tracer (FP and FI) concentrations remain constant in the 20 μL Complete Assay regardless of changes to other reaction conditions.
Figure 2. An Outline of the Procedure. The DDX5 Enzyme Reaction is initiated by the addition of yeast RNA and ATP. After the Enzyme Reaction incubation is completed, ADP detection reagents are added (Transcreener ADP2 Antibody and Tracer) along with EDTA to quench the DDX5 reaction.
10 μL Enzyme Reaction Components
| Components | Working Stock | Final Concentration in 10 µL | |
| Enzyme Assay Buffer D, 10X | 1X in Nuclease Free Water | 1X (50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 0.01% Triton X-100) | |
| DDX5 Enzyme, 0.1 mg/mL (1.91 µM) | 2X in 1X Enzyme Assay Buffer D | 12 nM – 20 nM* | |
| ATP, 5 mM | 200 µM in 1X Enzyme Assay Buffer D (with 2 mg/mL Yeast RNA) | 100 µM | |
| Yeast RNA, 10 mg/mL | 2 mg/mL in 1X Enzyme Assay Buffer D (with 200 µM ATP) | 1 mg/mL |
*See Section 4.1 for Determining the Optimal Enzyme Concentration.
Table 1. DDX5 Enzyme Reaction Components. Concentrations are provided for the standard protocol using 5 µL of DDX5 Enzyme Mix and 5 µL of RNA/ATP Substrate Mix for the Enzyme Reaction.
Table 2. 1X ADP Detection Mix Components. The optimal concentrations for each of the detection reagents based on the preferred readout mode are shown. Changes to the concentrations may require re-optimization of the assay.
Determining the Optimal Enzyme Concentration
Using the enzyme concentration suggested in the DDX5 Enzyme Certificate of Analysis should provide a robust signal that is within the linear range for ADP formation. However, for best results, we suggest performing an enzyme titration to identify the optimal enzyme concentration (EC50 to EC80), especially when running the assay in a different buffer system or with a different ATP or Yeast RNA concentration. This example uses a 2X serial dilution; it should be performed at least in duplicate. If a compound screen is planned, you should include the solvent (e.g., DMSO) at its final assay concentration.
4.1.1 Enzyme Titration Steps
1. Prepare 1000 µL 1X Enzyme Assay Buffer D: Dilute 100 µL of 10X Enzyme Assay Buffer D in 900 µL Ultrapure Nuclease Free Water.
2. Prepare 30 µL of 500 nM DDX5 Enzyme: dilute 7.85 µL of 1.91 µM DDX5 Enzyme in 22.15 µL 1X Enzyme Assay Buffer D.
3. Add 10 µL of the DDX5 Enzyme to well 1 (including replicates).
4. Add 5 μL of 1X Enzyme Assay Buffer D to wells 2-11, DO NOT add the Assay Buffer to well 1.
5. 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 DDX5 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.
6. Prepare 300 µL of RNA/ATP Substrate Mix: mix 60 µL of 10 mg/mL Yeast RNA and 12 µL of 5 mM ATP in 228 µL 1X Enzyme Assay Buffer D.
7. Start the Enzyme Reaction by adding 5 μL of the RNA/ATP 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 600 µL 1X ADP Detection Mix based on the concentrations provided in Table 2: 60 µL 10X Stop & Detect Buffer B, 6 µL ADP2 Alexa Fluor 633 Tracer and 109 µg/mL ADP2 Antibody in Ultrapure Nuclease Free Water.
Note: The 1X ADP Detection Mix varies for the readout mode used. Table 2 lists the 1X ADP Detection Mix for each readout.
9. Add 10 µL of 1X ADP 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 60 minutes before measuring signals.
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 a 50–80% change in signal (EC50 to EC80) (see Figure 3). This will result in initial velocity conditions, which correspond to the linear phase of the reaction after conversion of values to ADP 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 3. Enzyme Titration Curve. Example DDX5 Enzyme titration. The ideal range of enzyme concentrations is between EC50 and EC80; the specific concentration may vary depending on the enzyme lot.
Performing Single Compound Screening and Dose-Response Assays
4.2.1 Experimental Samples
1. Perform a serial dilution of test compounds 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 15 minutes) at room temperature to allow equilibration of the E-I complex.
Note: Final concentration of test compounds should be based on the volume of the Enzyme Reaction.
2. Start the Enzyme Reaction by adding 5 µL of the RNA/ATP Substrate Mix in each well. 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. After the incubation, add 10 µL of 1X ADP Detection Mix to the 10 µL Enzyme Reaction and mix the 20 µL Complete Assay using a plate shaker.
Note: The 1X ADP Detection Mix varies for the readout mode used. Table 2 lists the 1X ADP Detection Mix for each readout.
4. Incubate at room temperature for 60 minutes before measuring signals.
Figure 4. Dose-Response Curve. Example titration of probe inhibitors SU 3327, SPP 86, and Suramin in the presence of DDX5 Enzyme.
Setting Up a Standard Curve
Use of a standard curve for conversion of values to amount of ADP formed allows quantitative measurement of the enzyme activity and accurate IC50 determinations; it is not typically done for screening at single concentrations. The standard curve mimics the 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 percent ADP conversion.
In this example, a 12-point standard curve was prepared using the concentrations of ADP and ATP shown in the following Table. Commonly, 8- to 12-point standard curves are used. Here we describe preparation of a standard curve from 100 μM to 0.5 μM ADP, which encompasses the appropriate range for this assay.
| % Conversion | ATP (μM) | ADP (μM) | |
| 100 | 0 | 100 | |
| 50 | 50 | 50 | |
| 25 | 75 | 25 | |
| 15 | 85 | 15 | |
| 10 | 90 | 10 | |
| 8 | 92 | 8 | |
| 4 | 96 | 4 | |
| 2 | 98 | 2 | |
| 1 | 99 | 1 | |
| 0.5 | 99.5 | 0.5 | |
| 0.2 | 99.8 | 0.2 | |
| 0 | 100 | 0 |
Table 3. Concentration of ATP and ADP to prepare a 12-point standard curve.
4.3.1 Preparing the Standard Curve
1. Prepare 1X Enzyme Assay Buffer D.
2. Dilute 5 mM ATP and ADP to 100 μM in 1X Enzyme Assay Buffer D. The volume for both dilutions should be based on the total amount of volume to be utilized during preparation of the ATP/ADP percent conversion solutions.
3. Starting from 100 μM, add proportional values of ATP and ADP with the method of choice (i.e., Automatic Dispenser). For manual preparations, add reagents in separate microcentrifuge tubes to generate the desired percentage conversions utilized in the standard curve. An example of the dilution scheme can be found in Table 3. Once all dilutions are completed, add 10 μL of each solution to the respective wells.
4. Afterwards, add 10 μL of 1X ADP Detection Mix. The 1X ADP Detection Mix varies for the readout mode used. Table 2 lists the 1X ADP Detection Mix for each readout.
5. Finally, mix for 40 to 60 seconds. Incubate at room temperature for 60 minutes before measuring signals.
Figure 5. ADP Standard Curve. Standard curve using 1X ADP Detection Mix for FP readout as shown in Table 2 (109 μg/mL ADP2 antibody, 4 nM ADP2 Tracer).
Figure 6. Enzyme Titration Curve Converted to ADP Formed. Raw polarization signal (mP) is converted to ADP formed using a standard curve as described in Section 4.3. Only the linear portion of the graph is shown; interpolation was performed using GraphPad Prism.
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 what is of most interest when considering the suitability of an assay for HTS: the usable screening or “assay window.” It is a dimensionless coefficient for the quality of the screening window 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, thus the assay is an excellent assay. When running the DDX5 Assay, run the controls with and without enzyme (no test compound) to achieve final results. Use the following formula to determine Z’.
Figure 7. Z’ Measurement. Complete Assay is performed with and without DDX5 Enzyme (n=12). Z’ is then calculated based on the formula shown in Section 4.4.
Appendix
Using the Assay with Different Volumes and Plate Formats
| Component | Total Volume | Enzyme Reaction Volume | 1X ADP 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 |
Please check the working plate volumes from the manufacturer to ensure they are within the suggest volumes ranges of your plate.
Links to Applicable Application Notes
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