Enzolution® RIG-I ATPase Assay System

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Enzolution RIG-I ATPase Assay System Tech Manual

A comprehensive assay system for measuring the activity of RIG-I 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

Enzolution RIG-I ATPase Assay System

Name: Enzolution® RIG-I ATPase Assay System
SKU: 21385
Availability: InStock

The Enzolution RIG-I ATPase Assay System includes RIG-I (Retinoic acid-inducible gene I) enzyme, substrate, assay plates, and buffers required for a RIG-I enzymatic reaction. The Transcreener ADP² Assay (sold separately), a far-red competitive assay designed for HTS and inhibitor dose-response measurements, measures ADP produced to determine activity. Used together, the Enzolution RIG-I Assay System and Transcreener ADP² Assay provide all reagents required to screen and profile RIG-I inhibitors.

The Enzolution Assay System is a valuable tool to advance drug discovery efforts in innate immune system pathways. RIG-I is a critical sensor of RNA viral infections, recognizing double-stranded RNA, 4-triphosphylated RNAs, and other pathogen-associated molecular patterns (PAMPs) that indicate the presence of a virus. It then induces a Type I IFN response via the NFkB and IRF3 pathways.

The Transcreener ADP² Assay is available for separate purchase through BellBrook Labs. For ADP assay ordering information, please view Transcreener ADP² Assay. The Transcreener ADP² 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 ADP² Assay Kit.

A Complete Assay Solution to Measure RIG-I Activity

Enzolution RIG-I ATPase Assay System Schematic

Enzolution Assay System includes enzyme, substrate, & buffers for the RIG-I reaction. Transcreener’s ADP² Assay determines resulting ADP activity.

Learn More About The ADP Assay Enzolution RIG-I ATPase Assay System Tech Manual

Explore RIG-I in the Innate Immune Response with This Free eBook

What’s Included

Enzolution RIG-I FP & FI Assay System
	1K		10K
Component	Part#	Qty	Part#	Qty
Recombinant RIG-I, Active, Human	2291	10 µg	2299	2 X 50 µg
RIG-I dsRNA, 8.1 µM	2290	24 µL	2302	240 µL
Enzyme Assay Buffer C, 10X	2292	2 mL	2304	20 mL
DTT, 1M	2296	100 µL	2295	1 mL
Corning 384-Well Black Assay Plates (#4514)	2229	3-Pack (1,000+ Assays)	2297	30-Pack (10,000+ Assays)

Enzolution RIG-I TR-FRET Assay System
	1K		10K
Component	Part#	Qty	Part#	Qty
Recombinant RIG-I, Active, Human	2291	10 µg	2299	2 X 50 µg
RIG-I dsRNA, 8.1 µM	2290	24 µL	2302	240 µL
Enzyme Assay Buffer C, 10X	2292	2 mL	2304	20 mL
DTT, 1M	2296	100 µL	2295	1 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.

How Does The Procedure Work?

The RIG-I reaction is initiated by the addition of a dsRNA/ATP substrate mixture. After the enzyme reaction is complete, ADP detection reagents (Transcreener ADP² Antibody & Tracer) and EDTA are added. Read the plate with a compatible microplate reader.

Excellent Z’ Data Amenable to HTS

The Enzolution RIG-I Assay System used with the Transcreener ADP assay produces robust results amenable to HTS. Z’ measurement using optimized RIG-I assay conditions (n=16). Z’ = 0.85.

Measure ADP to Determine RIG-I Activity Under Initial Velocity Assay Conditions

Raw polarization signal (mP) is converted to ADP formed using a standard curve. Conversion of raw data shows assay linearity. Here, we use 100 µM ATP with 16.2 nM dsRNA substrate. The enzyme buffer included 50 mM Tris, 2.5 mM MgCl₂, 50 mM NaCl, 0.01% Brij-35, and 5 mM DTT. The 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 82 µg/mL ADP antibody. The Detection Mix was added and incubated for 60 minutes at room temperature.

Enzyme Titration – FP Readout

Linear Response

Screen and Profile RIG-I Inhibitors for Small Molecule Drug Discovery

Transcreener Assays are designed for screening compound libraries and obtaining dose responses in a high throughput format. Follow-up SAR can be performed using the assay to determine inhibitor potency with ease. Assay conditions included 100 μM ATP, 16.2 nM 19-bp-dsRNA oligomer, 50 mM Tris, 2.5 mM MgCl₂, 50 mM NaCl, 0.01% Brij-35, 5 mM DTT, 1X Stop & Detect Buffer B, 4 nM ADP² AlexaFluor 633 Tracer, and 82 μg/mL ADP² Antibody.

Dose Response Curve

IC₅₀ (BVT 948) = 3.56 μM
IC₅₀ (Suramin) = 6.20 μM

Three Fluorescent Detection Mode Options

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, 93.7 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 197.8 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

Recombinant Full-Length RIG-I Enzyme

Active, human Retinoic acid-inducible gene I enzyme (AA 1-925), full-length, 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 50 mM Tris, 500 mM NaCl, and 10% Glycerol (pH 8.0). The RIG-I protein is currently used with the Transcreener assay at a concentration of 1.31 nM in a 10 μL reaction.

Go to Human RIG-I Enzyme Page

RIG-I ATPase Profiling Services

Interested in moving your program forward with BellBrook’s RIG-I assay services? BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your RIG-I 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-I/MDA5
DDX3
CD39
CD73

Articles

Resources

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FAQ

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Publications

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Application Notes

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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

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

Enzolution RIG-I ATPase Assay System