Transcreener® ADP² DDX3 Assay

Learn More About Transcreener Method Used for This DDX3 Assay

Discover DDX3 Modulators with the Transcreener ADP2 DDX3 Assay

The Transcreener ADP² Assay directly measures the ADP produced by DDX3 (also known as DEAD-box RNA Helicase 3 or DDX3 Helicase). 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 therapeutics for a variety of different conditions including antiviral therapies, autoimmune diseases, and cancer.

The kit comes complete with all the detection reagents required to measure activity. DDX3 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.

The Role of DDX3 As Therapeutic Target

DDX3 is part of the DEAD-box group of proteins which are a family of RNA helicases. These proteins have an active role in various cellular activities including innate immunity, viral reproduction, and tumorgenesis (Kukhanova, 2020). 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.

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. The Transcreener ADP² Assay can then be used to measure the ADP produced in the reaction to measure enzymatic activity.

Direct Detection of ADP to Measure DDX3 Enzymatic Activity

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

ADP FI Kinase Assay

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

ADP TR-FRET ATPase Assay

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

Download Tutorial

Learn why direct ADP detection is the best HTS assay method for Kinases and ATPases

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    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

    The Transcreener ADP² DDX3 Assay is in a simple, HTS-ready, mix-and-read format. Run your enzyme reaction, add Transcreener reagents, and read your plates. The assay is compatible with 96, 384, and 1536-well formats.

    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

    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. Both reactions have 1 mM ATP, 10 µM dsRNA, and 30 nM DDX3 for an enzyme reaction of 60 minutes at 37°C. Z' shown here = 0.73.

    DDX3 Assay Z Prime Measurement

    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
    bioteklogo 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
    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

    Transcreener ADP2 DDX3 Assay Kits

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

    Assay Plates for FI & FP

    Assay Plates for TR-FRET

    Cat. No.Product DescriptionPrice 
    2234Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)$50.00Add to Cart
    2231Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)$60.00Add to Cart
    2232Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)$499.00Add to Cart

    BellBrook Labs
    5500 Nobel Drive, Suite 230
    Madison, Wisconsin 53711 USA
    (608) 443-2400

    info@bellbrooklabs.com

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