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Learn More About the Method Used for the NUDT5 Assay.

NUDT5 Assay - A Transcreener AMP2 Assay Application

Transcreener AMP² Assay directly measures AMP produced by NUDT5 (nudix hydrolase 5 or NUDIX5). These AMP measurements allow researchers to effectively determine the activity of the NUDT5 enzyme. The assay provides a powerful tool to screen compound libraries for NUDT5 modulators to help find new therapies for disease. 

The kit comes complete with the detection reagents required to measure activity. NUDT5 enzyme & ADPR is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme. 

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 This NUDT5 Assay Work?

The Transcreener NUDT5 Assay determines NUDT5 enzyme activity by directly measuring AMP formed by the enzyme using the Transcreener AMP2/GMP2 Assay. By detecting AMP output, the assay provides a universal method to assess the activity of any AMP-producing enzyme in real-time. 

Transcreener NUDT5 Assay technology uses a simple but highly effective method that consists of an antibody selective to AMP over a far-red fluorescent tracer. AMP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout. 

The NUDT5 assay is available with FP and TR-FRET. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS. 

Direct Detection of AMP to Measure NUDT5 Enzymatic Activity

Fluorescent Polarization (FP)

Schematic of NUDT5 Assay

The workhorse. Used in many large screens. Best deck and signal stability.

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

NUDT5 TR-FRET Readout Schematic

For customers who prefer TR-FRET detection. Uses the same filter set as HTRF®

Applications

  • Measure Enzymatic Activity of NUDT5
  • Screen Compound Libraries for NUDT5 Inhibitors
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling

Features

  • Direct detection of unlabeled AMP
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method
  • Available in FP or TR-FRET readouts

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

Run your enzyme reaction, add Transcreener reagents, and read your plates. The NUDT5 assay is compatible with 96, 384, and 1536-well formats. 

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized NUDT5 reaction conditions indicate a robust assay.  Quality data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Z' shown here = 0.91

NUDT5 Assay Z Prime Measurement

Detection of AMP Under NUDT5 Initial Velocity

The assay demonstrates linearity when raw data is converted to AMP using a standard curve. NUDT5 can use different substrates.  ADPR at 100 μM starting concentration is shown here under initial velocity conditions. The enzyme reaction occurred for 60 minutes at room temperature.

Human NUDT5 Enzyme Titration

NUDT5 Assay Enzyme Titration Data

Linear Response

Linear Response Data NUDT5

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.

Dose-Response

NUDT5 Assay Dose Response with TH5427

IC50 = 4.66 nM

NUDT5 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 NUDT5 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

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.

Far-Red FP & TR-FRET Readouts Validated on Major Multimode Readers

SupplierInstrumentFP AssaysTR-FRET Assays
TriStar2S LB 942In ReviewClick Me ArrowValidated
Tristar 5In ReviewIn Review
Mithras2 LB 943In ReviewClick Me ArrowValidated
CytationTM 5ValidatedValidated
CytationTM 3ValidatedValidated
CytationTM 1ValidatedValidated
SynergyTM H1ValidatedValidated
SynergyTM 2/H4/4Click Me ArrowValidatedClick Me ArrowValidated
SynergyTM HTXNot CapableNot Capable
SynergyTM Neo 2ValidatedValidated
POLARstar® OmegaClick Me ArrowValidatedClick Me ArrowValidated
FLUOstar® OmegaNot CapableClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidatedClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidatedClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidatedClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidatedClick Me ArrowValidated
SenseIn ReviewIn Review
Analyst® GT/HTClick Me ArrowValidatedClick Me ArrowValidated
Gemini® XPS/EMNot CapableNot Capable
SpectraMax® M2/M2eNot CapableNot Capable
SpectraMax® M5/M5e/FlexStation® 3Not CapableClick Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowValidatedClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidatedClick Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidatedClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidatedClick Me ArrowValidated
Infinite® M200Not CapableNot Capable
Infinite® F500Click Me ArrowValidatedClick Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact UsClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidatedClick Me ArrowValidated

What's Included

ComponentNotes
AMP2/GMP2 AntibodyAntibody specific to the nucleotides AMP and GMP. The antibody concentration used for the NUDT5 Assay is 8 μg/mL.
AMP2/GMP2 TracerWhen displaced, changes in fluorescence can be read in an FP or TR-FRET readout, depending on the assay format.
Tris SolutionUsed to buffer the detection mixture.
AMP and GMPAMP is used to create the ADPR/AMP Standard Curve

What You Will Need

ComponentNotes
NUDT5 EnzymeWe have successfully used recombinant human NUDT5 from R&D Systems Cat. #6414-NH-010.
Enzyme BufferEnzyme buffer components optimized for your target. See the technical manual for potential interfering components.
ADPRWe have successfully used starting ADPR concentrations between 1-100 μM ADPR from Sigma-Aldrich Cat. #A0752
Assay PlatesAssay Plates Can Be Purchased Directly Through BellBrook Labs

FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514

TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513
Plate ReaderA multi-detection microplate reader configured to measure the output of the AMP tracer is required.
Liquid Handling DevicesUse liquid handling devices that can accurately dispense sub-microliter 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.

Assay Plates for TR-FRET

The Role of NUDT5 As a Therapeutic Target

NUDIX hydrolases, a superfamily of enzymes, hydrolyze modified nucleoside diphosphates. NUDT1 is one of the most well-studied NUDIX hydrolases with correlation to several disorders including Parkinson's disease and cancer. NUDT5 recently attracted interest due to linkage to some human malignancies. NUDT5 hydrolyzes 8-OH-dGDP, 8-OH-dADP, and ADP-ribose (ADPR) along with other substrates.

High amounts of NUDT1 and NUDT5 correlate with non-small-cell lung cancer (NSCLC) cell lines and tissues, and greater levels of both proteins in NSCLC patients link to tumor metastasis. NUDT5 expression elevates in human breast cancer cell lines and breast tumor tissues. High NUDT5 expression profiles exhibit significantly poorer prognoses than lower NUDT5 levels.

The Transcreener AMP2 Assay is an excellent tool for researchers examining the therapeutic effects of NUDT5.

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