Enzolution® POLQ Helicase ATPase Assay System

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Enzolution POLQ Helicase ATPase Assay System Tech Manual

A comprehensive assay system for measuring the activity of POLQ ATPase Activity.

Includes enzyme, substrate, plates, and buffers
For use with the trusted Transcreener ADP² Assay (Sold Separately)
Easy-to-use and HTS-ready
Screen & perform dose-response assays quickly in your lab

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SKU: N/A Category: Enzyme Assay Systems Tag: ADP Assay

Enzolution POLQ Helicase ATPase Assay System

Name: Enzolution® POLQ Helicase ATPase Assay System
SKU: 21374
Availability: InStock

The Enzolution POLQ Helicase ATPase Assay System includes POLQ helicase (DNA polymerase theta) enzyme, substrate, assay plates, and buffers required for a POLQ enzymatic reaction. The Transcreener ADP Assay (sold separately), a far-red competitive assay designed for HTS and inhibitor dose-response measurements, measures ADP produced by ATP hydrolysis to determine POLQ activity.

POLQ is synthetically lethal with BRCA-1 and ATM mutations, and both the polymerase and ATPase activities are being targeted with small molecules for anti-cancer drug discovery. The Enzolution POLQ Assay System, when used with the Transcreener ADP Assay Kit, provides all reagents required to screen and profile POLQ 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 the Enzolution Assay System, it is important to choose the assay system readout corresponding to the FP, FI, or TR-FRET configuration of your Transcreener ADP Assay Kit.

The Comprehensive Approach to Measure POLQ Enzymatic Activity

Enzolution POLQ Helicase Assay System Schematic

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

Learn More About The ADP Assay Enzolution POLQ Helicase ATPase Assay System Tech Manual

Explore POLQ in the DNA Damage Response (DDR) in this Free eBook

Specifications

What’s Included

Enzolution POLQ FP & FI Assay System
	1K		10K
Component	Part#	Qty	Part#	Qty
Recombinant POLQ Helicase Enzyme, Active, Human	2287	2 μg	2289	20 μg
POLQ ssDNA, 40 μM	2286	60 μL	2288	600 μL
Enzyme Assay Buffer A, 10X	2278	2 mL	2281	20 mL
Corning 384-Well Black Assay Plates (#4514)	2229	3-Pack (1,000+ Assays)	2230	30-Pack (10,000+ Assays)

Enzolution POLQ TR-FRET Assay System
	1K		10K
Component	Part#	Qty	Part#	Qty
Recombinant POLQ Helicase Enzyme, Active, Human	2287	2 μg	2289	20 μg
POLQ ssDNA, 40 μM	2286	60 μL	2288	600 μL
Enzyme Assay Buffer A, 10X	2278	2 mL	2281	20 mL
Corning 384-Well White Assay Plates (#4513)	2231	3-Pack (1,000+ Assays)	2232	30-Pack (10,000+ Assays)

What You Will Need

Component	Notes
Transcreener ADP Assay	Available for separate purchase through BellBrook Labs. View Transcreener ADP Assay to learn more. Choose the readout configuration (FP, FI, or TR-FRET) that is the best fit based on preference and plate reader compatibility.
Ultrapure Nuclease Free 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.
Plate Reader	A multi-detection microplate reader configured to measure output of the ADP tracer is required.
Liquid Handling Devices	Use liquid handling devices that can accurately dispense submicroliter volumes into 384-well plates.

An Outline of the Complete Protocol

The POLQ enzyme reaction is initiated by the addition of DNA substrate and ATP. After the enzyme reaction is completed, ADP detection reagents are added (Transcreener ADP² Antibody and Tracer).

Robust Assay Yields Quality Data

The Enzolution POLQ Assay System used with the Transcreener ADP Assay produces robust results amenable to HTS. Z’ measurement using optimized POLQ assay conditions (n=12). Z’ = 0.93.

Detection of POLQ 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 100 nM POLQ ssDNA substrate. The enzyme buffer included 50 mM Tris (pH 7.5), 1 mM MgCl₂, and 0.01% Triton. The POLQ enzyme reaction took place for 60 minutes at 30°C. The ADP Detection Mix included 1X Stop & Detect Buffer B, 4 nM ADP² Alexa Fluor 633 Tracer, and 55 µg/mL ADP antibody. The Detection Mix was added and incubated for 60 minutes at room temperature.

POLQ Enzyme Titration

Linear Response

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 0.5 nM POLQ helicase, 50 μM ATP, 100 nM 30-bp-ssDNA oligomer, 50 mM Tris, 1 mM MgCl₂, 0.01% Triton (pH 7.5), 1X Stop & Detect Buffer B, 4 nM ADP² AlexaFluor 633 Tracer, and 55 μg/mL ADP² Antibody.

Pilot & Interference Screen

Dose Response Curve

IC₅₀ [CBA] = 1.56

IC₅₀ [Suramin] = 0.92

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

FI Detection Mix (10 μL): 1X Stop & Detect Buffer B, 50 μg/mL ADP² Antibody-IRDye QC-1, 8 nM ADP² AlexaFluor 594 Tracer.

TR-FRET

TR-FRET Detection Mix (10 μL): 1X Stop & Detect Buffer C, 8 nM ADP² Antibody-Terbium Conjugate, and 500 nM ADP HiLyte647 Tracer.

Overview of the Transcreener ADP Assay (Sold Separately)

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.

View More About the ADP Assay

BellBrook’s Recombinant POLQ Helicase Enzyme

Active, human DNA polymerase theta enzyme (AA 67-894), C-terminal 6xHis-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 20 mM HEPES, 5 mM NaCl, 5% Glycerol, and 0.5 mM TCEP (pH 7.5). Currently used with the Transcreener assay at a concentration of 0.42 nM in a 10 μL reaction.

Go to Human POLQ Helicase Enzyme Page

POLQ Helicase Assay Services

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

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 k_off 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 a Scientist About Services Today

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.

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

POLQ As a Therapeutic Target

POLQ helicase functions as an ssDNA-dependent ATPase that can unwind short DNA duplexes and displace replication protein A (RPA) from single-stranded DNA. It also acts as a scanning motor protein that searches for microhomologies in the break region, using the ssDNA break on the opposite side of the break as a template for DNA extension to fill the gap.

Normal adult tissues express low or no POLQ. However, it overexpresses in lung, stomach, breast, ovarian, small intestine, and colorectal cancers. In each of these tumor types, high expression of POLQ tightly correlates with decreased survival. Since an expanding range of cancers seems to become addicted to POLQ in response to the loss of more accurate DSB repair systems, there is an opportunity to extinguish such cancers by targeting POLQ. Clinical trials began with promising candidate therapeutics in early 2022.

Read more in this article from BellBrook Labs: Looking Ahead: POLQ in the Future of Immuno-Oncology

Articles

Resources

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

FAQ

Protocols

Publications

Instrument Compatibility

Application Notes

Posters

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.

“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.”

- BioPharma Lead Discovery Program Manager

“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.”

- Pharmaceutical Research Scientist

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

- Director Pharma Drug Discovery Center

“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.”

- Biotech Program Manager

“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.”

- University Research Scientist

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

- Biotech Research Scientist

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BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400

info@bellbrooklabs.com

Also required (not included):

Transcreener ADP² Kinase Assay Kits – FP, 1,000 Assay, 384 Well