Transcreener® ADP² PKR Assay

Learn More About Transcreener Method Used for this PKR Assay.

Transcreener ADP2 PKR Assay

The Transcreener ADP² Assay directly measures the ADP produced by PKR  (also known as Protein Kinase R, interferon-induced, double-stranded RNA-activated protein kinase, eukaryotic translation initiation factor 2-alpha kinase 2, or EIF2AK2). These ADP measurements allow researchers to effectively determine the activity of the PKR enzyme. The assay provides a powerful tool to screen compound libraries for PKR modulators to help find new therapies for disease.

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

The Role of PKR As a Therapeutic Target

Protein Kinase R is a serine-threonine kinase with a central role in anti-viral activity through various PKR pathways. PKR decreases viral replication by reducing the phosphorylation of eukaryotic translation initiation factor (eIF2α), preventing further mRNA translation and protein synthesis. PKR also activates antiviral interferon cytokines through activation of nuclear factor kappa-light-chain-enhancers of activated B cells (NFkB) and induces apoptosis of viral cells, limiting spread of viral infection. The role PKR plays in anti-viral pathways has made it an emerging target for antiviral therapies. (Gal-Ben-Ari et al., 2019)

The Transcreener ADP² PKR Assay is an excellent tool for researchers examining the antiviral effects of PKR.

Direct Detection of ADP to Measure PKR Enzymatic Activity

Transcreener PKR Assay Schematic

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 PKR
    • Screen Compound Libraries for PKR 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 PKR 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 PKR Initial Velocity (eIF2α Substrate)- FP Readout

    The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 10 µM ATP with 200 nM eukaryotic initiation factor-2α (eIF2α). Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The enzyme buffer includes 50 mM Tris pH 7.5, 10 mM MgCl2, and 0.01% Brij. The Detection mix included 40 μg/mL ADP2 antibody, 4 nM ADP Alexa 633 tracer, and 1X Stop & Detect Buffer B in 10 μL. The PKR enzyme reaction took place for 60 minutes at room temperature. The Detection Mix was added and incubated for 40 minutes at room temperature and read with a CLARIOstar plate reader.

    PKR Enzyme Titration

    PKR Enzyme Titration elF2a

    Linear Response

    PKR Linear Curve

    Detection of ADP Under PKR Initial Velocity (MBP Substrate) - FP Readout

    The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 10 µM ATP with 100 ug/mL Myeline Basis Protein (MBP). Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The enzyme buffer includes 50 mM Tris pH 7.5, 10 mM MgCl2, and 0.01% Brij. The Detection mix included 40 μg/mL ADP2 antibody, 4 nM ADP Alexa 633 tracer, and 1X Stop & Detect Buffer B in 10 μL. The PKR enzyme reaction took place for 60 minutes at room temperature. The Detection Mix was added and incubated for 40 minutes at room temperature and read with a CLARIOstar plate reader. 

    PKR Enzyme Titration

    PKR Enzyme Titration MBP

    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 320 ng/mL PKR, 50 nM Tris, 0.01% Brig, 10 mM MgCl2, 100 μM ATP, 200 nM eIF2α . The Detection Mix consists of 1X Stop & Detect Buffer B, 40 μg/mL ADP Ab, and 4 nM 633 tracer. Assay was inhibited with Staurosporine for 30 minutes prior to the enzymatic reaction at room temperature. 

    Dose-Response - Staurosporine

    Staurosporine Dose Response in PKR Assay

    IC50 = 0.086 µM

    Robust Assay Yields Quality Data

    Z’ measurements using optimized PKR 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 = 0.91. Assay conditions based off eIF2α as substrate. 

    PKR Robust Assay

    Three Fluorescent Readouts Available

    Choose the readout that is the best fit for your lab based on preference and plate reader compatibility.

    Fluorescent Intensity

    PKR Fluorescent Intensity

    FI Detection Mix (10 μL): 1x Stop & Detect Buffer B, 40 μg/mL ADP Ab-IRDye QC1, 8 nM AlexaFluor 594 Tracer. Assay conditions based off eIF2α as substrate. 

    TR-FRET

    TR-FRET Detection Mix (10 μL): 1x Stop & Detect Buffer  C, 8 nM ADP Ab-Tb, 1000 nM ADP HiLyte647 tracer. Assay conditions based off eIF2α substrate. 

    PKR 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 PKR 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

    Transcreener ADP2 PKR 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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