PARG Assay System

Interrogating CD38 & PARG with Transcreener's ADPR Assay

PARG Assay System

The PARG Assay System includes PARG (poly (ADP-ribose) glycohydrolase) enzyme, polyADPR, assay plates, and buffers required for a PARG reaction to produce ADPR. The Transcreener ADPR Assay (sold separately) uses coupling enzyme to convert ADPR to AMP, which is detected using far-red tracers that minimize interference.

PARG plays an important role in DNA damage repair and has been seen as a potential target for anticancer therapy. The PARG Assay System, when used with the Transcreener ADPR Assay Kit, provides all reagents required to screen and profile PARG modulators.

The Transcreener ADPR Assay is not included in the assay system but is available for separate purchase through BellBrook Labs. For ADPR assay ordering information, please view Transcreener ADPR Assay.

The Comprehensive Approach to Measure PARG Enzymatic Activity

PARG Assay System Schematic

Assay System includes enzyme, substrate, & buffers for the PARG reaction. The Transcreener ADPR Assay measures corresponding PARG activity.

An Outline of the Complete Protocol

The enzyme reaction is run in the presence of coupling enzyme, so that ADPR is converted to AMP in real time. After the enzyme reaction incubation is complete, AMP detection reagents are added along with EDTA to quench the coupling enzyme. 

Outline of the PARG Assay

Robust Assay Yields Quality Data

The PARG Assay System used with the Transcreener ADPR assay produces robust results amenable to HTS. Z' measurement using optimized PARG assay conditions (n=15). Z' = 0.87.

PARG Z' Data

Detection of PARG Under Initial Velocity

Conversion of raw data to ADPR formed using a standard curve demonstrates assay linearity. The enzyme buffer includes 50 mM Tris (pH 7.5), 10 mM MgCl2, 0.001% BSA, and 0.01% Brij-35. The PARG enzyme reaction took place for 60 minutes at 30°C. The Detection Mix consists of 1X Stop & Detect Buffer B, 2 µg/mL AMP2/GMP2 Antibody, and 8 nM AMP2/GMP2 Alexa Fluor 633 tracer. The Detection Mix was added and incubated for 90 minutes at room temperature and read with a CLARIOstar plate reader. 

PARG Enzyme Titration

PARG Assay Enzyme Titration Data

Linear Response

Linear Response Curve for PARG

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. Here, we used a known PARG probe inhibitor, PDD 00017273. Assay conditions included 50 mM Tris, 10 mM MgCl2, 0.001% BSA, 0.01% Brij-35, 8 nM AMP2/GMP2 AlexaFluor 633 Tracer, and 2 μg/mL AMP2/GMP2 Antibody.

Inhibitor Dose Response Profiling

PARG Dose Response with PDD 00017273

IC50 = 3 nM

Overview of the Transcreener ADPR Assay (Sold Separately)

Transcreener ADPR Assay Schematic Final

The Transcreener ADPR Assay is a biochemical HTS assay for quantitative measurement of ADP-ribose (ADPR) production in enzymatic reactions. The assay uses a coupling enzyme to convert ADPR into AMP, which is then detected using a far-red, competitive fluorescence polarization (FP) assay. 

The assay is designed for high throughput screening (HTS). The sensitivity and selectivity of the assay allows for the ability to obtain robust assay signals (>100 mP) with less than 10 nM of enzyme and a Z' value greater than 0.7. It is available in an easy-to-use, mix-&-read format. 

BellBrook's Recombinant PARG Enzyme

Active, full-length (aa 1-976), human PARG enzyme, C-terminal 6xHis-tagged protein expressed and purified from Baculovirus-infected insect cells. The enzyme has been thoroughly validated with Transcreener ADPR Activity Assays. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is >80% in a 50 mM Tris (pH 7.5), 500 mM NaCl, 10% glycerol, and 1 mM TCEP composition. Currently used with the Transcreener assay at a concentration between 5.3 - 6.1 pM in a 10 μL reaction.

PARG Enzyme Graphic

PARG Assay Services

Interested in moving your program forward with BellBrook's PARG Assay services? BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your PARG discovery 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.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.

Contact Us to Learn More

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What's Included

What You Will Need

PARG Assay System

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

Cat. No.Product DescriptionPriceAdd to Cart
3033-1KPARG Assay System (1,000 Assay, 384 Well)$989.00Add to Cart
3033-10KPARG Assay System (10,000 Assay, 384 Well)$4,449.00Add to Cart
3033-100KPARG Assay System (10 X 10,000 Assay, 384 Well)$19,989.00Add to Cart

Transcreener ADPR Assay Kits

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

Cat. No.Product DescriptionPriceAdd to Cart
3030-1KTranscreener ADPR FP Assay (1,000 Assay, 384 Well)$899.00Add to Cart
3030-10KTranscreener ADPR FP Assay (10,000 Assay, 384 Well)$4,999.00Add to Cart
3030-100KTranscreener ADPR FP Assay (10 X 10,000 Assay, 384 Well)$23,995.00Add to Cart

Related Resources

Interconnections Between the DNA Damage Response and Innate Immunity

There is extensive crosstalk between the DNA damage response (DDR) pathways and innate immune pathways. Both the individual pathways and the interconnections between them are a focus for exciting new small molecule drug therapeutics that target cancers and debilitating autoimmune disorder. The Transcreener HTS Assay platform accelerates these efforts by providing a robust and easy-to-use biochemical assay to measure activity of key enzymes in the innate immune and DDR pathway.

In this guide, we provide an overview of the DDR and innate immune pathway, and describe Transcreener Assays and Assay Systems for key therapeutic targets. The enzyme targets discussed include:

DDR:

  • POLQ
  • WRN
  • PARP1
  • PARG
  • DNA PK

Innate Immunity:

  • cGAS
  • TREX1
  • TBK1
  • IKKβ
  • IRAK4
  • ENPP1
  • CD38
  • OAS-1
  • RIG-1/MDA5
  • DDX3
  • CD39
  • CD73
DNA Damage Response and Innate Immunity Preview

PARG As a Therapeutic Target

PARG, along with PARP1, are the principal elements of the DNA damage response (DDR). When single-strand breaks occur in cellular DNA, PARP1 mediates the poly ADP ribosylation of itself and target proteins, such as histones, promoting the decompaction of chromatin and recruiting relevant enzymes to initiate DNA repair.

Eventually, PARP1 poly ADP ribosylates itself to the point of inactivity. Upon completion of the repair, PARG hydrolyzes poly (ADP-ribose) (PAR) on PARP1 and similarly tagged associated proteins to diminish intracellular PAR and disassemble the repair machinery at a given site. This also reactivates the previously heavily poly ADP ribosylated PARP1.

Learn more in this article: Discovering the Emerging Importance of PARG in Immunity

Related Resources

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Related Webinars

[Webinar] Targeting DNA Damage Repair for Drug Discovery Using the Transcreener ADPR® Assay

Preview of DNA Damage Repair Webinar

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

info@bellbrooklabs.com

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