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. 

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

Supplier Instrument FP Assays
TR-FRET Assays
bioteklogo TriStar²S LB 942 in review validated
Mithras² LB 943 in review validated
bioteklogo Cytation™ 5 validated validated
Cytation™ 3 validated validated
Cytation™ 1 validated validated
Synergy™ H1 validated validated
Synergy™ 2/H4/4 validated validated
Synergy™ HTX not capable not capable
Synergy™ Neo 2 validated validated
BMGLABTECH Logo POLARstar® Omega validated validated
FLUOstar® Omega not capable validated
PHERAstar® FSX validated validated
PHERAstar® Plus/FS validated validated
CLARIOstar® /Plus validated validated
MDS AT logo Analyst® GT/HT validated validated
Gemini® XPS/EM not capable not capable
SpectraMax® M2/M2e not capable not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable validated
SpectraMax® Paradigm validated contact us
SpectraMax® iD3/iD5 in review validated
perkinElmerLogo EnVision®/EnVision® Xcite validated validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated validated
Infinite® M200 not capable not capable
Infinite® F500 validated validated
Infinite® F200/Ultraevolution contact us contact us
Spark™ 10M validated validated

What's Included

What You Will Need

Transcreener AMP2 Assay Kits - For NUDT5 Activity Assays

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

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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