Interrogating CD38 & PARG with the Transcreener ADPR Assay

ADPR Assay Methods

Ordering Information

ADPR Assay Services

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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)

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

Learn More About the ADPR Assay in this Poster

Transcreener ADPR Assay Tech Manual

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.

Nanomolar Sensitivity and Outstanding Selectivity

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.

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.

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 K_M (0.82 μM) or saturating (10 μM) concentrations. The enzyme titration shows the assay response of PARG at saturating substrate.

PARG Enzyme Titration

Linear Response

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

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

CD38 Assay System Page

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Far Red FP Readouts Validated on Major Multimode Readers

Supplier	Instrument	FP Assays
	TriStar²S LB 942	In Review
	Tristar 5	In Review
	Mithras² LB 943	In Review

	Cytation^TM 5	Validated
	Cytation^TM 3	Validated
	Cytation^TM 1	Validated
	Synergy^TM H1	Validated
	Synergy^TM 2/H4/4	Validated
	Synergy^TM HTX	Not Capable
	Synergy^TM Neo 2	Validated

	POLARstar^® Omega	Validated
	FLUOstar^® Omega	Not Capable
	PHERAstar^® FSX	Validated
	PHERAstar^® Plus/FS	Validated
	CLARIOstar^® /Plus	Validated
	VANTAstar^TM	Validated

	Sense	In Review

	Analyst^® GT/HT	Validated
	Gemini^® XPS/EM	Not Capable
	SpectraMax^® M2/M2e	Not Capable
	SpectraMax^® M5/M5e/FlexStation^® 3	Not Capable
	SpectraMax^® Paradigm	Validated
	SpectraMax^® iD3/iD5	Validated
	SpectraMax^® i3x	Validated

	EnVision^®/EnVision^® Xcite	Validated

	Infinite^® M1000/M1000Pro/Safire2^TM	Validated
	Infinite^® M200	Not Capable
	Infinite^® F500	Validated
	Infinite^® F200/Ultra^evolution	Contact Us
	Spark^TM 10M	Validated

What's Included


Component	Notes
AMP²/GMP² Antibody	Sufficient antibody is included in the kit to complete 1,000 assays or 10,000 assays. 1.26 mg/mL solution in PBS with 10% glycerol.
AMP²/GMP² AlexaFluor 633 Tracer	AlexaFluor^® 633 labeled tracer in 2 mM HEPES (pH 7.5), 0.01% Brij-35. The final tracer concentration in the 20 μL complete assay is 4 nM. When displaced, changes in fluorescence can be read in an FP format.
ADPR-AMP Coupling Enzyme	Coupling enzyme in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM DTT, 10% glycerol.
Stop & Detect Buffer B, 10X	The Stop & Detect Buffer B will stop the coupling enzyme by chelating Mg²⁺. To ensure that the reaction is completed stopped, confirm that the EDTA concentration is at least equimolar to the magnesium ion concentration in the reaction. The final concentration of Stop & Detect Buffer B at the time of FP measurement is 0.5X.
ADPR	The ADPR in this kit can be used to create a standard curve to convert mP values to ADPR product formed.

What You Will Need


Component	Notes
Enzyme	Transcreener 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.
Substrate	The 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 Plates	Assay 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
Plate Reader	A microplate reader configured to measure FP is required for the assay.
Liquid Handling Devices	Use liquid handling devices that can accurately dispense submicroliter volumes into 384-well plates.
Ultrapure Water	Some 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.

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Transcreener ADPR Assay Kits

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

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

Assay Plates for FP

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

CD38 Assay System

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

Cat. No.	Product Description	Price	Add to Cart
3031-1K	CD38 Assay System (1,000 Assay, 384 Well)	$549.00	Add to Cart
3031-10K	CD38 Assay System (10,000 Assay, 384 Well)	$2,289.00	Add to Cart
3031-100K	CD38 Assay System (10 X 10,000 Assay, 384 Well)	$9,160.00	Add to Cart

CD38 Enzyme Ordering Information

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

Cat. No.	Product Description	Price	Add to Cart
2274	Recombinant CD38 Enzyme, Active, Human, 1 µg	$299.00	Add to Cart
2275	Recombinant CD38 Enzyme, Active, Human, 10 µg	$598.00	Add to Cart

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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.

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 k_off 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.

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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.