Learn More About Transcreener Method Used for this PKR Assay.

PKR Assay - A Transcreener ADP2 Assay Application

Transcreener ADP² Assay directly measures ADP produced by PKR  (Protein Kinase R 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.

How Does This PKR Assay Work?

The Transcreener PKR Assay determines PKR 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 PKR Assay 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 PKR 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 PKR Enzymatic Activity

Fluorescent Polarization (FP)

Transcreener PKR Assay Schematic

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 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

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

Transcreener Mix and Read Assay

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

Detection of ADP Under PKR Initial Velocity (eIF2α Substrate)

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 with eIF2a substrate

Linear Response

PKR Linear Curve

Detection of ADP Under PKR Initial Velocity (MBP Substrate)

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

Three Fluorescent Readout Options

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

Fluorescent Intensity

PKR Assay with FI Readout Data

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

PKR Assay with TR-FRET Readout Data

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

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 via 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.

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