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Cutting-Edge Assay Solutions for DNA Damage Repair & Innate Immunity Targets

Enzolution™ DDX5 ATPase Assay System

The Enzolution DDX5 Assay System is a comprehensive assay technology to assess activity of DDX5, a protein that plays a pivotal role in innate immunity by acting as a viral RNA sensor.

  • Enzyme, substrate, plates, and buffers
  • For use with the trusted Transcreener ADP2 Assay (Sold Separately)
  • Easy-to-use and HTS-ready
  • Screen & perform dose-response assays quickly in your lab

A Complete Assay Solution Ready to Tackle DDX5 Discovery

The Enzolution DDX5 Assay System provides researchers with an assay tool that will simplify and advance their exploration of novel DDX5/innate immunity modulators. The assay system includes DDX5 enzyme, RNA substrate, assay plates, and buffers to produce a DDX5 enzymatic reaction. 

The Enzolution Assay System is intended for use with the trusted Transcreener ADP2 Assay (sold separately). The Transcreener ADP2 Assay is the only commercially available ADP assay that directly detects ADP production, i.e. without the use of coupling enzymes. The direct detection format minimizes compound interference, producing trustworthy results with Z' values greater than 0.7. The assay is an extensively validated ADP detection method used in millions of wells by researchers globally.

When purchasing the Enzolution Assay System, it is important to choose the assay system readout that corresponds to the FP, FI, or TR-FRET configuration of your Transcreener ADP2 Assay.

DDX5 ATPase Assay System Schematic
DNA Damage Response and Innate Immunity Preview

Download the free eBook, The DNA Damage Response and Innate Immunity, to explore Transcreener Assays for DNA Damage Repair & Innate Immunity.

A Simple Two-Step Protocol

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.

DDX5_Assay_Outline

Robust HTS-Ready Assay

Z' of 0.92 confirms the robustness of the DDX5 assay. A robust system like this, is necessary to ensure the precision and efficiency required to scale-up assays for high-throughput screening.

Z' Data using DDX5 Assay

Direct Detection of DDX5 Activity with the Trusted Transcreener ADP2 Assay

Transcreener assays have fewer reagents and a less complex mechanism than any other nucleotide detection assay, which generally require coupling enzymes to convert a nucleotide to a product that can generate a signal with a reporter enzyme. The direct detection Transcreener format decreases the risk of false positives or missing a hit.

Transcreener Assay Overview

BellBrook's Recombinant DDX5 Enzyme (Included in Assay System)

Active, human DDX5 (AA 40-475) protein expressed and purified from Baculovirus. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is >80% in a composition of 50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM TCEP, and 10% Glycerol (pH 7.5). Currently used with the Transcreener assay at a concentration of 22.46 nM in a 10 μL reaction.

DDX5 Enzyme Graphic

DDX5 Activity Under Initial Velocity Conditions

Here, we use 100 µM ATP with 1 mg/mL Yeast RNA substrate. The enzyme buffer included 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, and 0.01% Triton X-100. The enzyme reaction took place for 60 minutes at 30°C. The ADP Detection Mix included 1X Stop & Detect Buffer B, 4 nM ADP2 Alexa Fluor 633 Tracer, and 109 µg/mL ADP antibody. The Detection Mix was added and incubated for 60 minutes at room temperature.

Enzyme Titration - FP Readout

DDX5 Enzyme Titration with FP Readout

Linear Response

Linear Response with DDX5 Assay

Simplify Your Discovery of Novel Innate Immunity Modulators

1280 compounds were screened from the Tocris 2.0 Library set. Selected hits from the pilot screen (SU 3327 and SPP 86) and the control compound were tested in dose-response mode.

Dose Response Mode

Dose Response with DDX5 Assay

IC50 (SU 3327) = 0.45 μM

IC50 (SPP 86) = 0.12 μM

IC50 (Suramin) = 6.94 μM

Choose the Fluorescent Detection Mode That Best Suits Your Preference

Choose the readout configuration that is the best fit for your lab based on preference and plate reader compatibility. The assay is available with an FP, FI, or TR-FRET readout. Reference our Instrument Compatibility resource to determine the detection mode compatible with your plate reader. 

Fluorescent Intensity

FI Titration with DDX5

TR-FRET

TR-FRET Titration with DDX5

Let Us Do The Work: DDX5 Assay Services

Interested in moving your DDX5 program forward, but don't want the hassle of bringing an assay in-house? BellBrook's expert scientists have you covered. Our team of biochemical and enzymology expert scientists will work diligently to progress your lead candidates through the early-stages of drug discovery. The best part - we are a small custom shop. We can customize assay conditions to suit your project needs, produce results faster, and you will directly collaborate with one of our expert scientists. 

BellBrook Scientist Performing Lead Discovery Services

Lead Discovery Services Include:

  • Inhibitor Screening – To identify or confirm activity with the target.
  • Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC50 results.
  • Residence Time Measurements Determination of koff using 'jump dilution' enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.
  • Triaging Non-stoichiometric Inhibitors – Run assays under varying conditions to weed out poor performers early.
  • Evaluate Compound-Target Binding – Run thermal shift assays to confirm compound-target engagement.

Contact a BellBrook Scientist Today

Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.

What's Included

What You Will Need

Enzolution DDX5 ATPase Assay System

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

Please choose the assay system readout corresponding to the FP, FI, or TR-FRET configuration of your Transcreener ADP Assay Kit.

Transcreener ADP Assay Kits

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

Related Resources

Development of HTS Enzymatic Assays for RNA Helicaes: DDX3, DDX5, DDX17, RIG-I, and MDA5

RNA helicases are a broad class of enzymes that bind and modify RNA in an ATP-dependent manner and play diverse roles in RNA metabolism, regulation of gene expression, and innate immunity. Several RNA helicases, especially in the dead box helicase (DDX) family, have been shown to be dysregulated in cancer, and there have been limited efforts to develop small molecule inhibitors. To accelerate development of selective helicase inhibitors while avoiding off target effects, especially with helicases that promote tumor immunity, we are assembling a panel of high throughput biochemical assays using the Transcreener ADP2 Assay for homogenous detection of RNA-dependent ATPase activity with a far-red fluorescence polarization (FP) readout. Here, we:

  • Developed assays for DDX3, DDX5 and DDX17, which have all been implicated in cancer, and the Rig-I-like receptor (RLR) helicases, RIG-I and MDA5, which trigger expression of type I interferons important for tumor immunity
  • Produced the recombinant enzymes using BaV-infected insect cells, as full-length polypeptides, or in some cases truncated to remove disordered N-terminal domains
  • Determined RNA substrate and cofactor requirements and kinetic parameters with the purified enzymes and optimized the assays for initial velocity detection of RNA-dependent ATPase activity with a Z’ value greater than 0.7, ensuring a sufficient signal window for inhibitor screening and dose response assays
  • Validated each of the helicase ATPase assays for HTS by screening a collection of bioactives (Tocris 2.0) and confirming hits in dose response mode with the enzymatic assay and with thermal shift assays to confirm target binding
RNA Helicases Poster Preview
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