Transcreener® ADPR Assay Kits
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Transcreener ADPR FP 1K Assay Kit

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Transcreener ADPR Assay Tech Manual
Detection of ADPR for measuring enzymatic activity for enzymes such as CD38, PARG, and SARM1
  • Screening for enzyme inhibitors/activators
  • Generating dose-response curves and IC50 values for inhibitors
  • Kinetic and mechanistic analyses
  • HTS-ready assay to speed up your discovery

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Overview – Transcreener ADPR Assay Kits

The Transcreener ADPR Assay is a biochemical assay that measures the production of ADPR (also known as ADP-ribose) in enzymatic reactions. It is designed specifically for high throughput screening (HTS), with a single-addition, mix-and-read format. As examples, this assay can be used to detect the activity of CD38 or PARG, both of which may have implications for several therapeutic indications.

The ADPR assay is easy to integrate into automated HTS workflows, with outstanding reagent stability, sensitive detection of ADPR down to 10 nM and as high as 50 μM, and compatibility with commonly used multimode plate readers.

The kit comes complete with detection reagents required to measure activity. Enzyme targets are not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme. CD38 enzyme and screening kits are available for separate purchase through BellBrook Labs.

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 the Transcreener ADPR Assay Work?

The Transcreener ADPR Assay measures enzymatic activity by using a coupling enzyme to convert ADPR into AMP. AMP is then detected using a proven Transcreener Assay format. By detecting ADPR in a homogenous format, the assay can be used with virtually any enzyme that produces ADPR.

Transcreener ADPR technology uses an antibody selective to AMP and a far-red fluorescent tracer. As AMP is produced, it competes with the tracer changing the fluorescent properties and providing a fluorescent readout. The assay uses a far-red tracer to minimize interference from fluorescent compounds and light scattering.

The Transcreener ADPR Assay is available with an FP fluorescent readout. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagents, and measure. The simplicity of the system yields robust results that also make it extremely amiable to HTS.

Transcreener ADPR Assay Schematic
Far Red FP Readouts Validated on Major Multimode Readers

Interrogating CD38 & PARG with the Transcreener ADPR Assay

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Specifications

What’s Included

ADPR FP Assay
1K10K
ComponentPart #QtyPart #Qty
AMP2/GMP2 AlexaFluor® 633 Tracer, 800 nM2097100 µL21011 mL
AMP2/GMP2 Antibody, 1.2 mg/mL2114158 µL23441.6 mL
Stop & Detect Buffer B, 10X20271 mL203210 mL
ADPR Coupling Enzyme, 400X226730 µL2269300 µL
5 mM ADPR226650 µL2268500 µL

What You Will Need

ComponentNotes
EnzymeTranscreener Assays are designed for use with purified enzyme preparations. Contaminating enzymes, such as phosphatases or nucleotidases, can produce background signal and reduce the assay window.
SubstrateThe Transcreener ADPR Assay is designed for use with purified enzymes capable of producing ADPR from a substrate. Substrate impurities and degradation may produce background signal and reduce the assay window.
Assay PlatesAssay Plates Can Be Purchased Separately From BellBrook Labs.

FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates (Cat. #4514) or 1536-well plates (Cat. #3728).
Plate ReaderA microplate reader configured to measure FP is required for the assay.
Liquid Handling DevicesUse liquid handling devices that can accurately dispense submicroliter volumes into 384-well plates.
Ultrapure WaterSome deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem.

Applications

  • Measure Enzymatic Activity of ADPR-Producing Enzymes

  • Screen Compound Libraries for Enzyme Modulators

  • Quantify Inhibitor Potency

  • Inhibitor Selectivity Profiling

  • Measure Drug-Target Residence Time

Features

  • Easy to use, mix-&-read, homogenous format
  • Sensitive detection of ADPR from 10 nM – 50 μM
  • Robust assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method

Easy-to-Use, Mix-&-Read, HTS-Ready Format

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

ADPR Outline of Protocol

Nanomolar Sensitivity and Outstanding Selectivity

ADPR Competition Curve

Competition curves show detection of ADPR as low as 10 nM and up to 50 μM and illustrate the ability to tune the dynamic range by adjusting AMP²/GMP² antibody concentration.

ADPR-AMP Coupling Enzyme Competition Curve

Titration curves show outstanding selectivity of coupling enzyme for ADPR vs. NAD+, polyADPR, and other nucleotides.

Obtain Accurate Dose Response Data Fast

Example dose response data for CD38. The Transcreener ADPR Assay allows inhibitor screening and profiling with accurate & quick results.

Dose Response Curve with CD38 Assay

Robust Assay Yields Quality Data

Z’ measurement using optimized CD38 assay conditions (n = 16). A Z’ of 0.93 demonstrates a robust assay method amenable to HTS.

CD38 Assay Produces Robust Data

Detection of ADPR to Measure PARG Activity

Assay demonstrates linearity when raw data is converted to ADPR using a standard curve. Assay can be run at both the substrate KM (0.82 μM) or saturating (10 μM) concentrations. The enzyme titration shows the assay response of PARG at saturating substrate.

PARG Enzyme Titration

PARG Enzyme Titration Using ADPR Assay

Linear Response

Linear Response ADPR Assay with PARG

Overview of BellBrook’s ADPR-Related Assay Systems (Sold Separately)

ADPR-Related Assay Systems Schematic

BellBrook Labs also offers Assay Systems, which includes enzyme, substrate, assay plates, and buffers required for an enzyme reaction to produce ADPR. This provides a comprehensive method to measure enzymatic activity when used with the Transcreener ADPR Assay. The Assay Systems and Transcreener ADPR Assay are sold separately.

Available ADPR-Related Assay Systems:

CD38 Assay System
PARG Assay System

ADPR 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 program.

Assay 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.
Scientist pipetting into a microplate beside a microscope.

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CD38 & PARG As Drug Targets

CD38 contributes to immune response, cell adhesion, and signal transduction. Furthermore, it plays a vital role in calcium signaling, acting as a second messenger to regulate muscle contraction, cell growth, fertilization, and cellular motility. Because of this role, CD38 is a target for tumor evasion and inflammatory-related diseases (BellBrook Labs, 2021).

PARG, along with PARP1 are the principal elements of the DNA damage response (DDR). A recent study showed that elevated levels of PARG reduced early tumorigenesis potential without decreasing cell viability. Because of this, PARG has become a target of interest for anticancer therapy (BellBrook Labs, 2022).

The Transcreener ADPR Assay allows researchers to screen compound libraries for CD38 or PARG modulators. This is an excellent tool to discover novel therapeutic treatments.

Related Resources

Resources

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

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