Enzolution® eIF4A3 ATPase Assay System

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Enzolution eIF4A3 ATPase Assay System Technical Manual

A comprehensive assay system for measuring the activity of eIF4A3.

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

Is your plate reader compatible? Find out now!

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SKU: N/A Category: Enzyme Assay Systems

Enzolution eIF4A3 Assay System

Name: Enzolution® eIF4A3 ATPase Assay System
SKU: 30883
Availability: InStock

The Enzolution eIF4A3 Assay System provides researchers with an assay tool that will simplify and advance their exploration of novel eIF4A3 modulators. The assay system includes eIF4A3 enzyme, RNA substrate, assay plates, and buffers to generate an eIF4A3 enzymatic reaction.

The Enzolution Assay System is intended for use with the trusted Transcreener ADP² Assay (sold separately). The Transcreener ADP² Assay is the only commercially available ADP assay that directly detects ADP production, i.e. without the use of coupling enzymes. The direct detection format minimizes compound interference, producing trustworthy results with Z’ values greater than 0.7. The assay is an extensively validated ADP detection method used in millions of wells by researchers globally.

When purchasing the Enzolution Assay System, it is important to choose the assay system readout that corresponds to the FP, FI, or TR-FRET configuration of your Transcreener ADP² Assay.

A Complete Assay Solution Ready to Tackle eIF4A3 Discovery

eIF4A3-ATPase-Assay-System-Schematic-1024x204

Need Protocol Help? Download Technical Manual Here

Poster: The DNA Damage Response and Innate Immunity

Specifications

What’s Included

FP & FI Assay System
	1K		10K
Component	Part#	Qty	Part#	Qty
Recombinant eIF4A3, Active, Human	2385	70 µL	2386	704 µL
Yeast RNA, 10 mg/mL	2319	200 µL	2311	1.4 mL
Enzyme Assay Buffer E, 10X	2320	2 mL	2321	20 mL
TCEP, 0.5M	2387	25 µL	2388	250 µL
Corning 384-Well Black Assay Plates (#4514)	2229	3-Pack (1,000+ Assays)	2230	30-Pack (10,000+ Assays)

TR-FRET Assay System
	1K		10K
Component	Part#	Qty	Part#	Qty
Recombinant eIF4A3, Active, Human	2385	70 µL	2386	704 µL
Yeast RNA, 10 mg/mL	2319	200 µL	2311	1.4 mL
Enzyme Assay Buffer E, 10X	2320	2 mL	2321	20 mL
TCEP, 0.5M	2387	25 µL	2388	250 µL
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.
Laboratory Incubator	An incubator model that is capable of maintaining temperature stability at 30°C is required.

A Simple Two-Step Protocol

The eIF4A3 Enzyme Reaction is initiated by the addition of yeast RNA and ATP. After the Enzyme Reaction incubation is completed, ADP detection reagents are added (Transcreener ADP² Antibody and Tracer) along with EDTA to quench the eIF4A3 reaction.

Robust HTS-Ready Assay

Z’ of 0.84 confirms the robustness of the eIF4A3 assay. A robust system like this, is necessary to ensure the precision and efficiency required to scale-up assays for high-throughput screening.

Direct Detection of eIF4A3 Activity with the Trusted Transcreener ADP² Assay

Transcreener assays have fewer reagents and a less complex mechanism than any other nucleotide detection assay, which generally require coupling enzymes to convert a nucleotide to a product that can generate a signal with a reporter enzyme. The direct detection Transcreener format decreases the risk of false positives or missing a hit.

BellBrook’s Recombinant eIF4A3 Enzyme (Included in Assay System)

Active, human, full-length eIF4A3 protein expressed and purified from E.coli. 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 >85% in a composition of 50 mM Tris-HCl, 150 mM NaCl, 0.5 M L-Arginine, 10% Glycerol, pH 8.0.

Explore the eIF4A3 Enzyme

eIF4A3 Activity Under Initial Velocity Conditions

Here, we use 30 µM ATP with 50 µg/mL Yeast RNA substrate. The enzyme buffer included 50 mM Tris-HCl (pH 7.5), 0.25 mM MgCl₂, 1 mM TCEP, and 0.01% Triton. The enzyme reaction took place for 120 minutes at 30°C. The ADP Detection Mix included 1X Stop & Detect Buffer B, 4 nM ADP² Alexa Fluor 633 Tracer, and 33.4 µg/mL ADP antibody. The Detection Mix was added and incubated for 60 minutes at room temperature and read on a plate reader.

Activity may vary from lot to lot. Please see C of A for enzyme activity.

Enzyme Titration – FP Readout

Linear Response

Simplify Your Discovery of Novel Innate Immunity Modulators

1280 compounds were screened from the Tocris 2.0 Library set. An interference screen was performed to eliminate compounds interfering with detection reagents. A selected hit from the pilot screen (L-798106) and the control compound were tested in dose-response mode.

Dose Response with Polygodial

eIF4A3 Enzolution Assay System Dose-Response Curve with Polygodial

IC₅₀ (Polygodial) = 29.05 μM

Dose Response with

Suramin

IC₅₀ (Suramin) = 4.311 μM

Dose Response with L-798, 106

eIF4A3 Enzolution Assay System Dose-Response Curve with L-798, 106

IC₅₀ (L-798, 106) = 20.16 μM

Choose the Fluorescent Detection Mode That Best Suits Your Preference

Choose the readout configuration that is the best fit for your lab based on preference and plate reader compatibility. The assay is available with an FP, FI, or TR-FRET readout. Reference our Instrument Compatibility resource to determine the detection mode compatible with your plate reader.

Fluorescent Intensity

TR-FRET

Let Us Do The Work: eIF4A3 Assay Services

Interested in moving your eIF4A3 program forward, but don’t want the hassle of bringing an assay in-house? BellBrook’s expert scientists have you covered. Our team of biochemical and enzymology expert scientists will work diligently to progress your lead candidates through the early-stages of drug discovery. The best part – we are a small custom shop. We can customize assay conditions to suit your project needs, produce results faster, and you will directly collaborate with one of our expert scientists.

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.
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 varying conditions to weed out poor performers early.
Evaluate Compound-Target Binding – Run thermal shift assays to confirm compound-target engagement.

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

Development of HTS Enzymatic Assays for RNA Helicases: DDX3, DDX5, DDX17, RIG-I, and MDA5

To accelerate development of selective helicase inhibitors while avoiding off target effects, especially with helicases that promote tumor immunity, we are assembling a panel of high throughput biochemical assays using the Transcreener ADP² Assay for homogenous detection of RNA-dependent ATPase activity with a far-red fluorescence polarization (FP) readout. Here, we:

Developed assays for DDX3, DDX5 and DDX17, which have all been implicated in cancer, and the Rig-I-like receptor (RLR) helicases, RIG-I and MDA5, which trigger expression of type I interferons important for tumor immunity
Produced the recombinant enzymes using BaV-infected insect cells, as full-length polypeptides, or in some cases truncated to remove disordered N-terminal domains
Determined RNA substrate and cofactor requirements and kinetic parameters with the purified enzymes and optimized the assays for initial velocity detection of RNA-dependent ATPase activity with a Z’ value greater than 0.7, ensuring a sufficient signal window for inhibitor screening and dose response assays
Validated each of the helicase ATPase assays for HTS by screening a collection of bioactives (Tocris 2.0) and confirming hits in dose response mode with the enzymatic assay and with thermal shift assays to confirm target binding

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