PKR Assay – Transcreener Application

Application Note: Detection of Protein Kinase R Activity with the Transcreener ADP2 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.

This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.

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 Kit

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 Kit

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. 

Z' Data for PKR

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 60 minutes at room temperature and read with a CLARIOstar plate reader.

PKR Enzyme Titration

PKR Assay Enzyme Titration with elF2a Substrate

Linear Response

PKR Linear Response Curve with elF2a substrate

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 60 minutes at room temperature and read with a CLARIOstar plate reader. 

PKR Enzyme Titration

PKR Assay Enzyme Titration with MBP Substrate

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

PKR Assay Dose Response Curve in Percent Inhibition

IC50 = 0.09 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

FI Readout for PKR Titration

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 Titration with a TR-FRET Readout

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

SupplierInstrumentFP AssaysFI AssaysTR-FRET Assays
TriStar2S LB 942In ReviewClick Me ArrowValidatedClick Me ArrowValidated
Tristar 5In ReviewClick Me ArrowValidatedIn Review
Mithras2 LB 943In ReviewClick Me ArrowValidatedClick Me ArrowValidated
CytationTM 5ValidatedValidatedValidated
CytationTM 3ValidatedValidatedValidated
CytationTM 1ValidatedValidatedValidated
SynergyTM H1ValidatedValidatedValidated
SynergyTM 2/H4/4Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SynergyTM HTXNot CapableValidatedNot Capable
SynergyTM Neo 2ValidatedValidatedValidated
POLARstar® OmegaClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
FLUOstar® OmegaNot CapableClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SenseIn ReviewClick Me ArrowValidatedIn Review
Analyst® GT/HTClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Gemini® XPS/EMNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M2/M2eNot CapableClick Me ArrowValidatedNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not CapableClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowValidatedClick Me ArrowContact UsClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidatedIn ReviewClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M200Not CapableClick Me ArrowValidatedNot Capable
Infinite® F500Click Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact UsClick Me ArrowValidatedClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidatedClick Me ArrowValidatedClick Me ArrowValidated

What's Included

What You Will Need

Transcreener ADP2 Assay Kits - For PKR Activity Assays

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

Cat. No.Product DescriptionPriceAdd to Cart
3013-ATranscreener ADP2 FI Assay (200 Assay, 96 Well)$359.00Add to Cart
3013-1KTranscreener ADP2 FI Assay (1,000 Assay, 384 Well)$759.00Add to Cart
3013-10KTranscreener ADP2 FI Assay (10,000 Assay, 384 Well)$4,299.00Add to Cart
3013-100KTranscreener ADP2 FI Assay (10 X 10,000 Assay, 384 Well)$21,495.00Add to Cart
3010-1KTranscreener ADP2 FP Assay (1,000 Assay, 384 Well)$725.00Add to Cart
3010-10KTranscreener ADP2 FP Assay (10,000 Assay, 384 Well)$4,099.00Add to Cart
3010-100KTranscreener ADP2 FP Assay (10 X 10,000 Assay, 384 Well)$20,495.00Add to Cart
3011-1KTranscreener ADP2 TR-FRET Red Assay (1,000 Assay, 384 Well)$789.00Add to Cart
3011-10KTranscreener ADP2 TR-FRET Red Assay (10,000 Assay, 384 Well)$4,449.00Add to Cart
3011-100KTranscreener ADP2 TR-FRET Red Assay (10 X 10,000 Assay, 384 Well)$22,245.00Add to Cart

Assay Plates for FI & FP

Cat. No.Product DescriptionPriceAdd to Cart
2233Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)$55.00Add to Cart
2229Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)$60.00Add to Cart
2230Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)$479.00Add to Cart

Assay Plates for TR-FRET

Cat. No.Product DescriptionPriceAdd to Cart
2234Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)$55.00Add to Cart
2231Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)$65.00Add to Cart
2232Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)$529.00Add to Cart

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.

Related Resources

Related Posts
Transcreener ADP Kinase Assay Reviews

BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400

info@bellbrooklabs.com

TOP