Enzolution® SIRT3 Assay System

Download Tech Manual

Enzolution SIRT3 Assay System Technical Manual

A comprehensive assay system for measuring the activity of SIRT3.

Includes enzyme, substrate, plates, and buffers
For use with the Transcreener OAADPr SIRT Assay (Sold Separately)
Easy-to-use, HTS-ready, SIRT3 screening assay
Screen & perform dose-response assays quickly in your lab
Profile against other Sirtuins, including SIRT1 and SIRT2

Is your plate reader compatible? Find out now!

Readout

Data Points

Clear

SKU: 3052 Category: Enzyme Assay Systems Tag: SIRT3 Assay

Enzolution SIRT3 Assay System

Name: Enzolution® SIRT3 Assay System
SKU: 3052
Availability: InStock

The Enzolution SIRT3 Assay System provides researchers with an assay tool to simplify and advance their exploration of novel SIRT3 modulators. The assay system includes SIRT3 enzyme, acetylated peptide, assay plates, and buffers to generate a SIRT3 enzymatic reaction.

The Enzolution SIRT3 Assay System is intended for use with the Transcreener OAADPr SIRT Assay Kits (Parts #3046 and #3047) to measure the enzymatic activity of SIRT3 (NAD dependent deacetylase sirtuin 3). SIRT3 catalyzes transfer of acetyl groups from lysine residues to NAD, yielding the unacetylated peptide, O-acetyl-ADP ribose (OAADPr), and nicotinamide as products. The assay relies on a Coupling Enzyme (CE) to convert OAADPr into AMP, which is then detected using a far-red, competitive fluorescence polarization (FP) or time-resolved Förster-resonance-energy-transfer (TR-FRET) assay. The assay has been optimized and extensively validated for high throughput screening (HTS) and inhibitor dose response measurements using most multimode plate readers.

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

A Complete Assay Solution Ready to Tackle SIRT3 Discovery

Need Protocol Help? Download Technical Manual Here

Learn How to Screen SIRTs Quickly and Effectively

Download the free poster, Homogenous Detection of O-Acetyl-ADP-ribose as a Generic HTS Assay Method for SIRTs, recently presented at AACR 2025.

What’s Included

FP Assay System
	1K		10K
Component	Part#	Qty	Part#	Qty
Recombinant SIRT3 Enzyme, Active, Human	2367	20 µg	2368	2 x 100 µg
Acetylated Peptide, 10 mM	2336	100 µL	2337	1 mL
Enzyme Assay Buffer G, 10X	2338	2 mL	2339	20 mL
MnCl₂, 1 M	2303	30 µL	2304	300 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 SIRT3 Enzyme, Active, Human	2367	20 µg	2368	2 x 100 µg
Acetylated Peptide, 10 mM	2336	100 µL	2337	1 mL
Enzyme Assay Buffer G, 10X	2338	2 mL	2339	20 mL
MnCl₂, 1 M	2303	30 µL	2304	300 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 OAADPr SIRT Assay	Available for separate purchase through BellBrook Labs. View Transcreener OAADPr SIRT Assay to learn more. Choose the readout configuration (FP 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 AMP 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 SIRT3 Enzyme Reaction is initiated by the addition of H3K9Ac peptide, NAD⁺ and CE. After the Enzyme Reaction incubation is completed, AMP detection reagents are added (Transcreener AMP²/GMP² Antibody and Tracer) along with EDTA to quench the enzyme reactions.

Transcreener OAADPr SIRT Assay FP Outline

Robust HTS-Ready Assay

Z’ of 0.96 confirms the robustness of the Enzolution SIRT3 assay system. A robust system like this is necessary to ensure the precision and efficiency required to scale up assays for high-throughput screening. No need to scale up on your own!

BellBrook’s Recombinant SIRT3 Enzyme (Included in Assay System)

Active, human SIRT3 (AA 101-399) protein expressed and purified from E.Coli with an N-Terminal His tag. The enzyme has been thoroughly validated with the Transcreener OAADPr Assay Kit. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is 95% in a composition of 50 mM HEPES, 200 mM NaCl, 1 mM TCEP, 1 mM EDTA, 10% Glycerol, pH 7.5.

Explore the SIRT3 Enzyme

SIRT3 Activity Under Initial Velocity Conditions

Data was generated using the Transcreener OAADPr SIRT FP Assay, with the Enzolution SIRT3 Assay System. The following conditions were used. Assay buffer G with 0.5 mM MnCl₂, 50 µM NAD+, 100 μM H3K9Ac Peptide, with 1X OAADPr coupling enzyme mix. The enzyme was added to the CE/substrate mix and allowed to incubate for 60 minutes. The 1X AMP detection mix was added and incubated for 120 minutes, and the plates were read via a plate reader.

Enzyme Titration – FP Readout

Linear Response

Simplify Your Discovery of Novel Innate Immunity Modulators

Compounds were screened from the Tocris 2.0 Library set. An interference screen was performed to eliminate compounds interfering with detection reagents. Interference less than 0.1%

Dose-Response with 3-TYP

IC₅₀ = 27.17 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 or TR-FRET readout. Reference our Instrument Compatibility resource to determine the detection mode compatible with your plate reader.

FP

TR-FRET

Let Us Do The Work: SIRT3 Assay Services

Interested in moving your SIRT3 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.
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.

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.

Frequently Asked Questions

Does the assay yield a high Z′-factor?

When used with Transcreener OAADPr SIRT Assay Kits, our Enzolution SIRT3 Assay System achieved a Z’-factor of 0.96, confirming the robustness of this assay system.

Can the assay isolate SIRT3-specific activity in the presence of other sirtuins or interfering enzymes?

No. You can not run multiple SIRT reactions at one time in a multiplexed format. However, you can profile against other SIRTs using additional Enzolution assay systems.

Is the Enzolution SIRT3 Activity Assay suitable for high-throughput screening (HTS)?

Absolutely. When used in combination with Transcreener OAADPr SIRT Assay Kits, it has been validated in 96-, 384-, and 1536-well plate formats with robust Z’ factors (>0.7), ensuring reproducibility and scalability for HTS campaigns.

What type of substrate does the assay use?

The Enzolution SIRT3 Assay System includes the substrate Acetylated Peptide, H3K9Ac. Other substrates may be used but have not been validated with the system.

Is the enzyme included in the kit?

Yes! SIRT3 enzyme is included with the Enzolution SIRT3 Assay System. Purchase the Transcreener OAADPr Assay and you have a complete package to screen for SIRT3 modulators in hours. Just bring your inhibitors.

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

info@bellbrooklabs.com

Transcreener® OAADPr SIRT Assay Kits

Enzolution SIRT3 Assay System