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Interrogating CD38 & PARG with the Transcreener ADPR Assay

Transcreener ADPR Assay

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.

Measure ADPR Production in Enzymatic Reactions with an FP Readout

Fluorescence Polarization (FP)

Transcreener ADPR Assay Schematic Final

Coupling enzyme converts ADPR into AMP. AMP tracer bound to AMP antibody is competed off with AMP produced, resulting in FP signal.

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

IC50 = 1.01 µM

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
PARG

Far Red FP Readouts Validated on Major Multimode Readers

SupplierInstrumentFP Assays
TriStar2S LB 942In Review
Tristar 5In Review
Mithras2 LB 943In Review
CytationTM 5Validated
CytationTM 3Validated
CytationTM 1Validated
SynergyTM H1Validated
SynergyTM 2/H4/4Click Me ArrowValidated
SynergyTM HTXNot Capable
SynergyTM Neo 2Validated
POLARstar® OmegaClick Me ArrowValidated
FLUOstar® OmegaNot Capable
PHERAstar® FSXClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidated
SenseIn Review
Analyst® GT/HTClick Me ArrowValidated
Gemini® XPS/EMNot Capable
SpectraMax® M2/M2eNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not Capable
SpectraMax® ParadigmClick Me ArrowValidated
SpectraMax® iD3/iD5Click Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidated
Infinite® M200Not Capable
Infinite® F500Click Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidated

What's Included

What You Will Need

Transcreener ADPR Assay Kits

* For custom or bulk orders (over 10,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

Assay Plates for FP

CD38 Assay System

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

CD38 Enzyme Ordering Information

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

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 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.

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.

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