Oct 24, 2025
Enzolution™ SIRT2 Assay System Technical Manual
- info 1
- 1BellBrook Labs LLC
- BellBrook LabsTech. support phone: +1 (608) 443-2400 email: [email protected]
Protocol Citation: info 2025. Enzolution™ SIRT2 Assay System Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zm91gqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 01, 2025
Last Modified: October 24, 2025
Protocol Integer ID: 228796
Keywords: enzolution sirt2 assay system, enzymatic activity of sirt2, sirt2 inhibitor, dependent protein deacetylase sirtuin, transcreener oaadpr sirt assay kit, sirt2, lysine residues to nad, full length human sirt2, coupling enzyme, unacetylated peptide, inhibitor dose response measurement, assay, enzymatic activity, nicotinamide as product, transfer of acetyl group, nicotinamide
Abstract
The Enzolution™ SIRT2 Assay System is intended for use with the Transcreener® OAADPr SIRT Assay Kits (Parts #3046 and #3047) to measure the enzymatic activity of SIRT2 (NAD-dependent protein deacetylase sirtuin 2). SIRT2 catalyzes transfer of acetyl groups from lysine residues to NAD, yielding the unacetylated peptide, O-acetyl-ADP ribose (OAADPr), and nicotinamide as products (Figure 1). 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.
The Enzolution SIRT2 Assay System provides all reagents required to screen and profile SIRT2 inhibitors when used with the Transcreener OAADPr SIRT Assay Kits, including purified, full length human SIRT2 (aa 1-389) and acetylated peptide, H3K9Ac (1-21). The protocol is configured for 384-well plates; use of different multi-well plate formats will require adjustment of reagent concentrations utilized in the assay.
Troubleshooting
Introduction
The Enzolution™ SIRT2 Assay System is intended for use with the Transcreener® OAADPr SIRT Assay Kits (Parts #3046 and #3047) to measure the enzymatic activity of SIRT2 (NAD-dependent protein deacetylase sirtuin 2). SIRT2 catalyzes transfer of acetyl groups from lysine residues to NAD, yielding the unacetylated peptide, O-acetyl-ADP ribose (OAADPr), and nicotinamide as products (Figure 1). 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.
The Enzolution SIRT2 Assay System provides all reagents required to screen and profile SIRT2 inhibitors when used with the Transcreener OAADPr SIRT Assay Kits, including purified, full length human SIRT2 (aa 1-389) and acetylated peptide, H3K9Ac (1-21). The protocol is configured for 384-well plates; use of different multi-well plate formats will require adjustment of reagent concentrations utilized in the assay.
Key Applications:
- Screening for SIRT2 inhibitors (or activators)
- Generating dose response curves and IC50 values for SIRT2 inhibitors
- Kinetic and mechanistic analyses
Figure 1. Schematic Overview of the Enzolution SIRT2 Assay System with the Transcreener OAADPr SIRT Assays. OAADPr produced by the target SIRT enzyme is completely converted to AMP in real time by the OAADPr Coupling Enzyme. In the detection step, the CE is quenched by EDTA, and AMP displaces a fluorescent tracer from the AMP2/GMP2 Antibody, resulting in decreased fluorescence polarization (A) or TR-FRET (B).
Product Specifications
| Product | Quantity | Part# | |
| Enzolution SIRT2 Assay System | 1,000 assays* | 3049-1K | |
| 10,000 assays* | 3049-10K |
*NOTE: The exact number of assays depends on enzyme reaction conditions. The kits are designed for use with 384-well plates, using a 20 µL complete assay volume.
Storage
Enzymes should be stored at -80°C; other reagents can be stored at –20°C. Though we have confirmed that enzyme reagents are stable and maintain greater than 80% activity up to 5 freeze-thaw cycles, we recommend aliquoting the reagents and snap-freezing for multiple uses to minimize loss of activity.
Use the reagents provided in this kit within 6 months from date of receipt
Materials Provided
| Component | Composition | Notes | |
| SIRT2 Enzyme | 0.5 mg/mL (11.36 μM)* in 50mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol, | Full-length protein (amino acids 1-389), C-Terminal His tag, 44.01 kDa. Sufficient enzyme is included in the kit to complete at least 1,000 assays (Part# 3049-1K) or 10,000 assays (Part# 3049-10K). | |
| H3K9Ac (1-21) | 10 mM in water | An acetylated peptide from the N-terminus of Histone H3, stored in RNAse Free Water; to be used at 100 μM in the SIRT2 reaction. | |
| Enzyme Assay Buffer G, 10X | 500 mM Tris (pH 7.5), 50 mM MgCl2, and 0.1% Triton X-100 | Enzyme Assay Buffer G, along with MnCl2 and DTT, has been optimized to support SIRT activity as well as CE activity. Changes to the assay buffer may affect SIRT enzyme activity and/or conversion of OAADPr to AMP. | |
| DTT, 1 M | 1 M in water | Add to 1X Enzyme Assay Buffer G to a final concentration of 1 mM. | |
| MnCl2, 1 M | 1 M in water | Add to 1X Enzyme Assay Buffer G to a final concentration of 0.5 mM. | |
| 384-Well Low Volume Assay Plates | Corning #4514 - FP and FI Only Corning #4513 – TR-FRET Only | Polystyrene non-binding surface assay plates in either a 3-pack (1,000+ Assays) or a 30-pack (10,000+ Assays). We strongly recommend the use of these plates as inconsistent results have been observed with other plates. |
*NOTE: The exact concentration may vary from batch to batch. Please refer to the Certificate of Analysis for an accurate concentration.
Materials Required but Not Provided
| Component | Notes | |
| Ultrapure Nuclease Free Water | Some deionized water systems are contaminated with enzymes that can degrade both nucleotide substrates and products, reducing assay performance. Use nuclease free water such as: Invitrogen Part # AM9930 | |
| Plate Reader | A multimode microplate reader configured to measure FP is required. Transcreener Assays have been validated on the following instruments: BioTek Synergy™2 and Synergy™4; BMG Labtech PHERAstar® Plus and CLARIOstar® Plus; Molecular Devices SpectraMax™ Paradigm; Perkin Elmer EnVision® and ViewLux; and Tecan Infinite® F500, Safire2™, and M1000. Full list of compatible plate readers and settings. | |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates. | |
| Laboratory Incubator | An incubator model that is capable of maintaining temperature stability at 30°C is required. |
Transcreener OAADPr SIRT FP Assay - SOLD SEPARATELY
| Component | Composition | Notes | |
| AMP2/GMP2 Antibody | 1.26 mg/mL solution in PBS with 10% glycerol | 1,000 assays (Part# 3046-1K) or 10,000 assays (Part # 3046-10K). | |
| AMP2/GMP2 Alexa Fluor 633 Tracer | 800 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35 | 1,000 assays (Part# 3046-1K) or 10,000 assays (Part# 3046-10K). | |
| Stop & Detect Buffer B, 10X | 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35 | The Stop & Detect Buffer B components quench the CE reaction by chelating metals required for activity. | |
| OAADPr Coupling Enzyme (CE) | 200X OAADPr Coupling Enzyme in 50 mM Tris (pH 7.5), 100 mM NaCl, 1 mM TCEP, 10% glycerol, 0.05% Triton X-100 | OAADPr Coupling Enzyme is present in excess to ensure OAADPr is completely converted to AMP. | |
| NAD+ | 5 mM in water | 1,000 assays (Part# 3046-1K) or 10,000 assays (Part# 3046-10K). | |
| AMP | 5 mM in water | Used for the AMP standard curve. | |
| Enzyme Assay Buffer G, 10X | 500 mM Tris (pH 7.5), 50 mM MgCl2, and 0.1% Triton X-100 | Enzyme Assay Buffer G, along with MnCl2 and DTT, has been optimized to support SIRT activity as well as CE activity. Changes to the assay buffer could affect SIRT enzyme activity and/or conversion of OAADPr to AMP. | |
| DTT, 1 M | 1 M in water | Add to 1x Enzyme Assay Buffer G to a final concentration of 1 mM. | |
| MnCl2, 1 M | 1 M in water | Add to 1X Enzyme Assay Buffer G to a final concentration of 0.5 mM. |
| Component | Composition | Notes | |
| AMP2/GMP2 Antibody-Tb | 800 nM solution in 25 mM HEPES buffered saline | 1,000 assays (Part# 3047-1K) or 10,000 assays (Part# 3047-10K) | |
| AMP2/GMP2 Hilyte 647 Tracer | 10 μM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35 | 1,000 assays (Part# 3047-1K) or 10,000 assays (Part# 3047-10K) | |
| Stop & Detect Buffer C, 10X | 500 mM HEPES, 0.2% Brij, and 200 mM EDTA at a final pH 7.5 | The Stop & Detect Buffer C components quench the CE reaction by chelating metals required for activity. | |
| OAADPr Coupling Enzyme (CE) | 200X OAADPr Coupling Enzyme in 50 mM Tris (pH 7.5), 100 mM NaCl, 1 mM TCEP, 10% glycerol, 0.05% Triton X-100 | OAADPr Coupling Enzyme is present in excess to ensure OAADPr is completely converted to AMP. | |
| NAD+ | 5 mM in water | 1,000 assays (Part# 3047-1K) or 10,000 assays (Part# 3047-10K) | |
| AMP | 5 mM in water | Used for the AMP standard curve. | |
| Enzyme Assay Buffer G, 10X | 500 mM Tris (pH 7.5), 50 mM MgCl2, and 0.1% Triton | Enzyme Assay Buffer G, along with MnCl2 and DTT, has been optimized to support SIRT activity as well as CE activity. Changes to the assay buffer could affect SIRT enzyme activity and/or conversion of OAADPr to AMP. | |
| DTT, 1 M | 1 M in water | Add to 1x Enzyme Assay Buffer G to a final concentration of 1 mM. | |
| MnCl2, 1 M | 1 M in water | Add to 1X Enzyme Assay Buffer G to a final concentration of 0.5 mM. |
Before You Begin
- Read the entire protocol and note any reagents or equipment needed (see Section 2.2).
- Check the plate reader and verify that it is compatible with the Transcreener OAADPr SIRT Assays
- Please read and understand the Transcreener OAADPr SIRT Assay Technical Manuals prior to use with this kit.
Protocol
The methods described below are for endpoint detection of OAADPr formation by SIRT2 under initial velocity conditions (≤ 10% conversion of NAD+ to OAADPr) with a sub-saturating concentration of NAD+ (50 μM) and H3K9Ac peptide (100 μM). These conditions will ensure sensitive detection of inhibitors that compete with either substrate. Significant changes in the NAD+ concentration may require adjustment of the dynamic range, as described in the Transcreener OAADPr SIRT Assay Tech Manuals (Section 3.2).
Figure 2. Simple Mix-and-Read Format. The SIRT Enzyme Reaction is run in the presence of CE, so that OAADPr is converted to AMP in real time. After the Enzyme Reaction incubation is complete, 1X AMP Detection Mix is added, which contains EDTA to quench the Enzyme Reaction. Plates are allowed to sit for 120 min at room temperature before reading to allow the detection reaction to reach equilibrium.
10 µL Enzyme Reaction Components:
| Component | Stock | Working Stock | Final Concentration in 10 µL | |
| Complete Assay Buffer | 10X Enzyme Assay Buffer G, 1 M DTT, 1 M MnCl2 | 1X Enzyme Assay Buffer G, 1 mM DTT, 0.5 mM MnCl2 in Nuclease Free Water | 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 0.5 mM MnCl2, 1 mM DTT, 0.01% Triton X-100 | |
| SIRT2 Enzyme | 0.5 mg/mL (11.36 μM) SIRT2 | 2X in Complete Assay Buffer | 8 nM – 30 nM (see Section 4.1) | |
| Substrate/CE Mix | 5 mM NAD+, 10 mM H3K9Ac peptide, 200X CE | 100 µM NAD+, 200 µM H3K9Ac peptide, 2X CE in Complete Assay Buffer | 50 µM NAD+, 100 µM H3K9Ac peptide, 1X CE |
Table 1. SIRT2 Enzyme Reaction Components.
Table 2. 1X AMP Detection Mix Components. The optimal concentrations for each of the detection reagents based on the preferred readout mode are shown. Changes to the concentrations may require re-optimization of the assay.
Performing an Enzyme Assay
The following assay protocol is for a 384-well format, using a 10 µL Enzyme Reaction and 20 µL Complete Assay volume when the plates are read. The use of different formats will require changes in reagent quantities (see Section 5.1). All the reagent mixes can be prepared ahead of time and stored on ice for at least 2 hours before use, with the exception of the Substrate/CE mix, which should be used within 30 minutes of preparation.
1. Prepare Working Stocks
a. Prepare Complete Assay Buffer by combining 1X Enzyme Assay Buffer G, 1 mM DTT, and 0.5 mM MnCl2 in Ultrapure Nuclease-Free Water.
b. Dilute SIRT2 enzyme to 2X the desired concentration* in Complete Assay Buffer.
c. Prepare Substrate/CE Mix by combining 2X CE, 100 µM NAD+, 200 µM H3K9Ac peptide in Complete Assay Buffer.
2. Run the SIRT2 Enzyme Reaction
a. Add 5 μL of 2X SIRT2 enzyme to each well, followed by 5 μL of Substrate/CE Mix to initiate the reaction. Mix gently on a plate shaker and incubate at 30°C for 60 minutes.
3. Prepare and Dispense AMP Detection Mix
a. Centrifuge AMP2/GMP2 Antibody at 10,000 x g for 10 minutes to remove any aggregates. It is normal for the antibody to form aggregates over time or after freeze/thaw cycles. Removing these aggregates will not affect assay performance.
b. Prepare 1X AMP Detection Mix by diluting Stop & Detect Buffer, AMP2/GMP2 Tracer, and AMP2/GMP2 Antibody in Ultrapure Nuclease-Free Water to the concentrations described in Table 2, according to the appropriate readout mode.
c. Add 10 μL of the 1X AMP Detection Mix to each well and mix gently on a plate shaker. Incubate at room temperature for 120 minutes to allow the detection reaction to reach equilibrium.
*Note: Each time an assay is performed with a new batch of enzyme, an enzyme titration is required to determine the working concentration.
*Using the EC80 concentration suggested in the SIRT2 Enzyme Certificate of Analysis should provide a robust signal that is within the linear range for AMP formation. However, for best results, it may be useful to perform an enzyme titration to identify the optimal enzyme concentration (EC50 to EC80) (see Figure 3a). The EC50 is provided by common graphing programs; the EC80 enzyme concentration can be calculated from the EC50, as follows:
ECX = (X ÷ (100 – X))(1 ÷ |hillslope| ) × EC50
Figure 3. Sample Data for FP Readout Mode. (a) Enzyme titration curve. (b) Raw polarization in enzyme titration curve is converted to AMP formed using the standard curve in Section 4.3. Only the linear portion of the graph is shown; interpolation was performed using GraphPad Prism (see Section 5.2 for guidance). (c) Dose-Response Curve of probe inhibitors (see Section 4.2). (d) Z’ Measurement (n=12) (see Section 4.4).
Performing Single Compound Screening and Dose-Response Assays
Single Compound Screening and Dose-Response Assays follow the protocol listed in Section 4.1. The target SIRT Enzyme is added to the test compounds pre-dispensed in wells; the total mixture volume should be 5 µL and DMSO concentration should not exceed 1-2%. We recommend mixing gently on a plate shaker for 40 to 60 seconds and preincubating for 30 minutes at room temperature to allow equilibration of the E-I complex.
NOTE: Final concentration of test compounds should be based on the volume of the Enzyme Reaction.
Setting Up a Standard Curve
Use of a standard curve for conversion of values to amount of AMP formed allows quantitative measurement of the enzyme activity and accurate IC50 determinations; it is not typically done for screening at single concentrations. The standard curve mimics the Enzyme Reaction and follows the protocol outlined in Section 4.1, with the SIRT enzyme replaced by a titration of AMP concentrations. The Substrate/CE Mix remains the same as in the SIRT Enzyme Reaction to account for background generated by NAD+ degradation and reaction with the CE.
Typically, an 8- to 12-point standard curve is used, with the starting concentration of AMP matching the NAD+ concentration in the SIRT Enzyme Reaction (see Figure 4). The AMP standard curve allows the calculation of the AMP concentration produced in the Enzyme Reaction and, consequently, the percent AMP conversion. An example protocol for a 12-point standard curve is shown below:
1. Prepare Working Stocks
a. Prepare Complete Assay Buffer and Substrate/CE Mix as described in Section 4.1.
b. Prepare 100 μM AMP in Complete Assay Buffer.
2. Perform AMP Titration
a. Add 5 μL of Complete Assay Buffer to well 2-12.
b. Add 10 μL of 100 μM AMP to well 1. Perform a 2-fold serial dilution by pipetting 5 μL of the AMP solution from well 1 to well 2, and so on. Well 12 should be kept as a blank.
c. Add 5 µl of Substrate/CE Mix, then mix gently on a plate shaker and incubate at 30°C for 60 minutes.
3. Prepare and Dispense AMP Detection Mix
a. Prepare the AMP Detection Mix as described in Section 4.1 and add 10 μL to each well. Mix gently on a plate shaker and incubate at room temperature for 120 minutes.
| Data Point | AMP (µM) | NAD+ (µM) | |
| 1 | 50 | 50 | |
| 2 | 25 | 50 | |
| 3 | 12.5 | 50 | |
| 4 | 6.25 | 50 | |
| 5 | 3.125 | 50 | |
| 6 | 1.563 | 50 | |
| 7 | 0.781 | 50 | |
| 8 | 0.391 | 50 | |
| 9 | 0.195 | 50 | |
| 10 | 0.098 | 50 | |
| 11 | 0.049 | 50 | |
| 12 | 0 | 50 |
Figure 4. AMP Standard Curves. Example concentration and data for a standard curve corresponding to 50 μM NAD+ in SIRT Enzyme Reaction.
Measuring Assay Robustness with Z'
The Z’ value is a dimensionless coefficient that quantifies the separation between the positive and negative controls, which is key to determining the robustness and reliability of an assay. A Z’ value of 0.5 or greater is typically considered indicative of a very good screening window for a biochemical assay, suggesting the assay is of excellent quality. To calculate the Z’ value, run the controls both with and without the enzyme (without test compounds). Then, use the following formula for the calculation:
Appendix
Using the Assay with Different Volumes and Plate Formats
Please check the working plate volumes from the manufacturer to ensure they are within the suggested volume ranges of your plate.
| Component | Total Volume | Enzyme Reaction Volume | 1X AMP Detection Mix Volume | |
| 96 Well Low Volume Plate | 50 µL | 25 µL | 25 µL | |
| 384 Well Low Volume Plate | 20 µL | 10 µL | 10 µL | |
| 1536 Well Low Volume Plate | 8 µL | 4 µL | 4 µL |
Links to Applicable Application Notes
Contact Information