Enzolution® CD38 Assay System
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Enzolution CD38 FP 1K Assay System

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Enzolution CD38 Assay System Tech Manual
The Enzolution CD38 Assay System is a comprehensive assay technology to assess the activity of CD38, a cancer and inflammatory-related disease target.
  • Includes enzyme, substrate, plates, and buffers
  • For use with the Transcreener ADPR Assay (Sold Separately)
  • Easy-to-use and HTS-ready
  • Ultra-sensitive detection – Use less enzyme!

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Enzolution CD38 Assay System

The Enzolution CD38 Assay System includes CD38 (Cluster of differentiation 38) enzyme, NAD+, assay plates, and buffers required for a CD38 reaction to produce ADPR. The Transcreener ADPR Assay (sold separately) uses coupling enzyme to convert ADPR to AMP, which is detected using a far-red, competitive assay to measure enzymatic activity.

CD38 inhibitors continue to be sought after to combat a plethora of illnesses, including cancer and inflammatory-related diseases. The Enzolution CD38 Assay System, when used with the Transcreener ADPR Assay Kit, provides all reagents required to screen and profile CD38 inhibitors.

The Transcreener ADPR Assay is not included in the Enzolution 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 CD38 Enzymatic Activity

Enzolution CD38 Assay System Schematic

Enzolution Assay System includes enzyme, substrate, plates & buffer for the CD38 reaction. Transcreener’s ADPR Assay measures ADPR produced.

Learn More About The ADPR Assay Enzolution CD38 Assay System Tech Manual

Interrogating CD38 & PARG with Transcreener’s ADPR Assay

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Specifications

What’s Included

Enzolution CD38 FP Assay System Assay
1K10K
ComponentPart #QtyPart #Qty
Recombinant CD38 Enzyme, Active, Human22741 µg22742 X 1 µg
NAD+, 5 mM227260 µL2273600 µL
Enzyme Assay Buffer B, 10X22702 mL227120 mL
Corning 384-Well Black Assay Plates (#4514)22293-Pack (1,000+ Assays)223030-Pack (10,000+ Assays)

What You Will Need

ComponentNotes
Transcreener ADPR AssayAvailable for separate purchase through BellBrook Labs. View Transcreener ADPR Assay to learn more.
Ultrapure Nuclease Free 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.
Plate ReaderA multi-detection microplate reader configured to measure FP of the AMP2/GMP2 Alexa Fluor 633 tracer is required.
Liquid Handling DevicesUse liquid handling devices that can accurately dispense submicroliter volumes into 384-well plates.

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.

Complete Outline of CD38 with ADPR Assay

Robust Assay Yields Quality Data

The Enzolution Assay System used with the Transcreener ADPR assay produces robust results amenable to HTS. Z’ measurement using optimized CD38 assay conditions (n=16). Z’ = 0.93.

CD38 Assay Produces Robust Data

Detection of CD38 Under Initial Velocity

Conversion of raw data to ADPR formed using a standard curve demonstrates assay linearity. The enzyme buffer included 50 mM Tris (pH 7.5), 10 mM MgCl2, 0.001% BSA, and 0.01% Brij-35. The CD38 enzyme reaction took place for 60 minutes at 30°C. The Detection Mix was added and incubated for 90 minutes at room temperature and read with a CLARIOstar plate reader. At 0.012 nM CD38, about 1.5 μM ADPR was produced, which was 10% substrate conversion from 15 μM NAD+ (approx. Km).

CD38 Enzyme Titration

Enzyme Titration for CD38 Assay

Linear Response

Linear Response Data with CD38

Screen for Inhibitors & Perform SAR

1280 compounds were screened from the Tocris 2.0 Library set. An interference screen was performed to eliminate compounds interfering with coupling enzyme and/or detection reagents. A total of 5 potential inhibitors were identified with polarization values ≥ 3 standard deviations above the mean. Out of these 5 compounds, one showed no interference with assay detection mixture and was selected for further study. The selected hit from the pilot screen was then tested in dose-response mode.

CD38 Pilot Screen

CD38 Pilot Screen of 1280 Small Molecules

Inhibitor Dose Response Profiling

Dose Response Curve with CD38 Assay

IC50 = 1.01 µM

Overview of the Transcreener ADPR Assay (Sold Separately)

Transcreener ADPR Assay Schematic

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.

View More About the ADPR Assay

BellBrook’s Recombinant CD38 Enzyme

Active, human Cluster of differentiation 38 enzyme, His-tagged protein expressed & purified. The enzyme is thoroughly validated with the Transcreener ADPR Assay Kit. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is >90% in a 50 mM Tris, 500 mM NaCl, 20% glycerol buffer composition (pH 8.0). Currently used with the Transcreener assay at concentrations between 0.1-1.2 nM.

Go to Human CD38 Enzyme Page
Graphic CD38 Enzyme

CD38 Assay Services

Interested in moving your program forward with BellBrook’s CD38 Assay services? BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your CD38 discovery 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.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.
Scientist pipetting into a microplate beside a microscope.

Contact Us About CD38 Assay Services

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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 disorders. 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

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The Role of CD38 As a Therapeutic Target

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.

Calcium levels are increased in patients with multiple myeloma (MM). MM is a type of cancer that affects plasma (white blood) cells, ultimately affecting antibody production. Additionally, asthma can result from calcium levels not being appropriately regulated. Discovering CD38 inhibitors will help combat a plethora of illnesses. Read more in this article from BellBrook Labs.

Related Resources

Resources

Find the right resources to help you get to your solution faster.

Link to: Frequently Asked Questions (FAQ’s)
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Link to: Technical Manuals
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Protocols
Link to: Publications
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Link to: Transcreener® Assays – Instrument Compatibility
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Link to: Application Notes
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Link to: Posters and Presentations
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Questions? Contact Us!

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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“Some CROs reserve their best teams for high profile customers. If you’re not right there with them you get less experienced teams… BellBrook understands enzyme kinetics, so it made that part of the project so much easier. The larger CRO didn’t seem to have the expertise we needed in this area.”

“The ADP assay is a great choice for measuring activity for any ADP-generating enzyme in both real time and endpoint formats. The universal detection system has been very useful for more challenging targets.”

“Excellent blend of simplicity & good science. Excellent technical support”

“The assay itself has good reproducibility and provides a clear readout on our enzyme activity. The most important piece, however, is the level of attention to our project and rapid turnaround time. The staff at BellBrook knows what they are doing.”

“The Transcreener AMP/GMP Assay is extremely straightforward to use and is very sensitive. In a UV-based enzyme assay I needed 1 µM enzyme to see a decent signal; however using the Transcreener AMP assay I only need 10 nM. Also, BellBrook Labs have been very helpful with the assay development. Their representative has been very willing to quickly help me get my assay up and running.”

“Through the process of developing and validating an assay for HTS applications, I generated a very nice data package of robust high quality data.”

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