Learn More About Transcreener Method Used for This DDX3 Assay

DDX3 Assay - A Transcreener ADP2 Assay Application

Transcreener ADP² Assay directly measures ADP produced by DDX3 (DEAD-box RNA Helicase 3). These ADP measurements allow researchers to effectively determine the activity of the DDX3 enzyme. The assay provides a powerful tool to screen compound libraries for DDX3 modulators to help find new therapies for disease. 

The kit comes complete with detection reagents required to measure activity. DDX3 enzyme is not included in the inhibitor screening assay kit. You can purchase DDX3 enzyme from BellBrook Labs separately.

How Does This DDX3 Assay Work?

The Transcreener DDX3 Assay determines DDX3 enzyme activity by directly measuring the ADP formed by the enzyme using the Transcreener ADP Assay. By detecting ADP output, the assay provides a universal method to assess the activity of any ADP-producing enzyme in real-time.

Transcreener DDX3 technology uses a simple but highly effective method that consists of an antibody selective to ADP over ATP and a far-red fluorescent tracer. ADP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.

The DDX3 assay is available with FP, FI, and TR-FRET. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.

Direct Detection of ADP to Measure DDX3 Enzymatic Activity

Fluorescent Polarization (FP)

The workhorse. Used in many large screens. Best deck and signal stability.

Fluorescent Intensity (FI)

ADP FI Kinase Assay

Positive FI signal. Compatible with simple fluorescence plate readers. Faster read time than FP or TR-FRET.

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

ADP TR-FRET Kinase Assay

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®.

Applications

  • Measure Enzymatic Activity of DDX3
  • Screen Compound Libraries for DDX3 Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Measure Drug-Target Residence Time

Features

  • Direct detection of unlabeled ADP
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method
  • Available in FP, FI, or TR-FRET readouts

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

Run your enzyme reaction, add Transcreener reagents, and read your plates. The DDX3 assay is compatible with 96, 384, and 1536-well formats. 

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized DDX3 reaction conditions indicate a robust assay.  Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Z' shown here = 0.73.

DDX3 Assay Z Prime Measurement

Detection of ADP Under DDX3 Initial Velocity - FP Readout

The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 500 µM ATP with 5 µM dsRNA substrate. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The buffer includes 25 mM Tris pH 7.5, 1 mM MgCl2 0.005% Brij, 225 μg/mL ADP2 antibody, and 2 nM ADP Alexa 633 tracer. The DDX3 enzyme reaction took place for 60 minutes at 37ºC in kinetic mode.

Human DDX3 Enzyme Titration

DDX3 Enzyme Titration Raw Data

Linear Response

Screen for Inhibitors & Perform SAR

Transcreener Assays are designed for screening compound libraries in a high throughput format. Follow-up SAR can also be performed using the assay to determine inhibitor potency with ease. Assay conditions include 15 nM DDX3 (BellBrook Labs), 500 µM ATP, and 5 µM dsRNA for 60 minutes at 37ºC in kinetic mode.

Dose-Response - Suramin

DDX3 Inhibition Assay with Suramin

IC50 = 0.092 µM

BellBrook Lab's Recombinant DDX3 Enzyme

Active, DEAD-box RNA helicase DDX3 enzyme (aa135-583), His-tagged protein expressed and purified from E.coli. The enzyme has been thoroughly validated with Transcreener ADP² Activity Assays. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is >90% in a 50 mM Tris-HCl pH 8.0, 150 mM NaCl, and 10% glycerol buffer composition. Currently used with the Transcreener assay at a concentration between 15 and 30 nM.

DDX3 Enzyme

DDX3 Assay Services

Interested in moving your program forward, but don't want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your DDX3 program.

Assay Development 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.

Contact Us to Learn More

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.

Far-Red FP, FI & TR-FRET Readouts Validated on Major Multimode Readers

Supplier Instrument FP Assays
FI Assays
TR-FRET Assays
berthold logo TriStar²S LB 942 in review validated validated
Mithras² LB 943 in review validated validated
bioteklogo Cytation™ 5 validated validated validated
Cytation™ 3 validated validated validated
Cytation™ 1 validated validated validated
Synergy™ H1 validated validated validated
Synergy™ 2/H4/4 validated validated validated
Synergy™ HTX not capable validated not capable
Synergy™ Neo 2 validated validated validated
BMGLABTECH Logo POLARstar® Omega validated validated validated
FLUOstar® Omega not capable validated validated
PHERAstar® FSX validated validated validated
PHERAstar® Plus/FS validated validated validated
CLARIOstar® /Plus validated validated validated
VANTAstar validated validated validated
hidex logo Sense in review validated in review
MDS AT logo Analyst® GT/HT validated validated validated
Gemini® XPS/EM not capable validated not capable
SpectraMax® M2/M2e not capable validated not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable validated validated
SpectraMax® Paradigm validated contact us contact us
SpectraMax® iD3/iD5 in review validated validated
perkinElmerLogo EnVision®/EnVision® Xcite validated validated validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated validated validated
Infinite® M200 not capable validated not capable
Infinite® F500 validated validated validated
Infinite® F200/Ultraevolution contact us validated contact us
Spark™ 10M validated validated validated

What's Included

What You Will Need

DDX3 Enzyme Ordering Information

* For custom or bulk orders (over 1,000 µg) please contact us (info@bellbrooklabs.com) for a quote.

Assay Plates for TR-FRET

The Role of DDX3 As a Therapeutic Target

DDX3 is a DEAD-box group protein (RNA helicase family). It is involved in several cellular activities, including innate immunity, viral reproduction, and tumorgenesis (Kukhanova, 2020). DDX3 unwinds various forms of dsRNA, both 5' - 3' and 3' - 5'. It requires ATP and Mg²+ to unwind and releases RNA via ATP hydrolysis.

In some viruses such as HIV, DDX3 enables viral replication, while in others (such as Dengue), it has an anti-viral effect. The RNA helicase has similar effects on some cancer studies, wherein one type DDX3 is a tumor suppressor, while in another it acts as an enhancer. These qualities make DDX3 an interesting target for treating a variety of diseases.

The Transcreener ADP2 DDX3 Assay is an excellent tool for researchers examining the therapeutic effects of DDX3.

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