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Transcreener ADP2 Assay for Universal ATPase/Helicase Screening & Profiling

Enzolution WRN Helicase ATPase Assay System

The Enzolution WRN Helicase ATPase Assay System includes WRN helicase (Werner syndrome helicase) enzyme, substrate, assay plates, and buffers required to measure WRN Helicase's ATPase activity. The Transcreener ADP Assay (sold separately), a far-red competitive assay designed for HTS and inhibitor dose-response measurements, measures ADP produced to determine enzymatic activity. 

WRN helicase inhibitors are sought-after therapeutics for targeting cancers that lack adequate mismatch repair machinery and enhanced mutation at microsatellite repeats (MSI-H). The Enzolution WRN Assay System, when used with the Transcreener ADP Assay Kit, provides all reagents required to screen and profile WRN helicase inhibitors.

The Transcreener ADP Assay is not included in the Enzolution Assay System but is available for separate purchase through BellBrook Labs. For ADP assay ordering information, please view Transcreener ADP Assay. The Transcreener ADP Assay is available with an FP, FI, or TR-FRET configuration. When purchasing an Enzolution Assay System, it is important to choose the assay system readout corresponding to the FP, FI, or TR-FRET configuration of your Transcreener Assay Kit.

The Comprehensive Approach to Measure WRN Enzymatic Activity

Enzolution WRN Helicase Assay System Schematic

Enzolution Assay System includes enzyme, substrate, & buffers for the WRN reaction. The Transcreener ADP Assay measures ADP produced.

An Outline of the Complete Protocol

The WRN helicase enzyme reaction is initiated by the addition of WRN-H-DNA substrate and ATP. After the enzyme reaction is completed, ADP detection reagents are added (Transcreener ADP2 Antibody and Tracer) along with EDTA to quench the WRN reaction.

WRN Helicase Assay Protocol Outline

Robust Assay Yields Quality Data

The Enzolution WRN Helicase ATPase Assay System used with the Transcreener ADP assay produces robust results amenable to HTS. Z' measurement using optimized WRN helicase assay conditions (n=16). Z' = 0.85.

Detection of WRN Helicase Under Initial Velocity - FP Readout

Conversion of raw data to ADP formed using a standard curve demonstrates assay linearity. Here, we use 50 µM ATP with 40 nM DNA substrate. The enzyme buffer included 50 mM Tris (pH 7.5), 1 mM MgCl2, and 0.01% Triton. The WRN helicase enzyme reaction took place for 60 minutes at 30°C. The ADP Detection Mix was added and incubated for 60 minutes at room temperature and read with a CLARIOstar plate reader. 

WRN Enzyme Titration

WRN Helicase Enzyme Titration

Linear Response

Linear Response Data with WRN Helicase Assay

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. Here, 1280 compounds were screened from the Tocris 2.0 Library set using the Transcreener ADP2 FP Assay. Assay conditions included 50 μM ATP, 100 nM DNA, 50 mM Tris, 1 mM MgCl2, 0.01% Triton (pH 7.5), 4 nM ADP2 AlexaFluor 633 Tracer, and 55 μg/mL ADP2 Antibody.

WRN Pilot Screen

Pilot Screen for WRN Helicase

Inhibitor Dose Response Profiling

Dose Response Data With WRN Helicase Assay

IC50 = 30 nM

Three Fluorescent Configurations Available

Choose the readout configuration that is the best fit for your lab based on preference and plate reader compatibility. 

Fluorescent Intensity

WRN Helicase Assay FI Data

FI Detection Mix (10 μL): 1X Stop & Detect Buffer B, 50 μg/mL ADP2 Antibody-IRDye QC-1, 4 nM ADP2 AlexaFluor 594 Tracer.

TR-FRET

TR-FRET Readout with WRN Helicase

TR-FRET Detection Mix (10 μL): 1X Stop & Detect Buffer C, 4 nM ADP2 Antibody-Terbium Conjugate, and 500 nM ADP HiLyte647 Tracer.

Overview of the Transcreener ADP Assay (Sold Separately)

ADP Assay Schematic

The Transcreener® ADP2 Assay is a far-red, competitive assay that measures ADP production to determine enzymatic activity. The technology uses a simple but highly effective method that consists of an antibody selective to ADP over ATP and a far-red fluorescent tracer. ADP produced in the reaction competes with the tracer, changing the fluorescent properties and providing a fluorescent readout.

The Transcreener® assay is designed specifically for high-throughput screening (HTS), with a single addition, mix-and-read format. It offers reagent stability and compatibility with commonly used multimode plate readers. The assay is available as an FP, FI, or TR-FRET configuration.

BellBrook's Recombinant WRN Helicase Enzyme

Active, human Werner syndrome helicase enzyme, His-tagged protein expressed and purified from insect cells. The enzyme has been thoroughly validated with the Transcreener ADP Assay Kit. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is >90% in a composition of 50 mM Tris-HCl, 500 mM NaCl, 20% Glycerol, 0.5 mM TCEP (pH 8.0). Currently used with the Transcreener assay at a concentration between 0.20 - 0.65 nM in a 10 μL reaction.

Horizontal Layout of WRN Helicase Enzyme Graphic

WRN Helicase Assay Services

Interested in moving your program forward with BellBrook's WRN helicase assay services? BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your WRN helicase discovery program.

Scientist Studying DDX41

ATPase Profiling 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.
  • Triaging Non-stoichiometric Inhibitors – Run assays under different conditions such as varying detergent and enzyme concentrations.
  • Evaluate Compound-Target Binding – Run thermal shift assays to confirm compound-target engagement via shifts in melting temperature.

Contact Us to Learn More

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What's Included

What You Will Need

Enzolution WRN Helicase ATPase Assay System

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

Please choose the assay system readout corresponding to the FP, FI, or TR-FRET configuration of your Transcreener ADP Assay Kit.

Transcreener ADP Assay Kits

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

Related Resources

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

WRN Helicase As a Therapeutic Target

WRN is one of the 5 evolutionarily conserved RecQ helicases found in humans. All human RecQ helicases hydrolyze ATP, unwind double stand DNA breaks, and are proficient at repairing DNA breaks. WRN helicase is capable of unwinding DNA duplexes, possessing at least 10 nucleotides of either a 3' or 5' single stranded DNA overhang, and is necessary for localizing the site of telomere damage. 

Cancers that lack adequate mismatch repair machinery and enhanced mutation at microsatellite repeats (MSI-H) depend upon active WRN for their survival. The helicase function of WRN seems to enable this subset of colorectal, endometrial, and gastric cancers to persist, in spite of their diminished DNA repair ability. The search for effective WRN helicase inhibitors offers hope to those who suffer from these MSI-H cancers. 

Read more in this article from BellBrook Labs: Can WRN Helicase Inhibitors Treat MSI-H Cancers?

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