Oct 20, 2025
Transcreener® OAADPr SIRT FP Assay Technical Manual
- info 1
- 1BellBrook Labs LLC
- BellBrook LabsTech. support phone: +1 (608) 443-2400 email: [email protected]
Protocol Citation: info 2025. Transcreener® OAADPr SIRT FP Assay Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkynr1g5r/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: September 29, 2025
Last Modified: October 20, 2025
Protocol Integer ID: 228473
Keywords: OAADPr SIRT FP Assay, transcreener oaadpr sirt fp assay, activity of human sirt1, human sirt1, sirt2, acetylated peptides as substrate, using acetylated peptide, biochemical ht, dependent deacetylase, sirt, assay, coupling enzyme, sirtuin, competitive fluorescence polarization, oaadpr into amp
Abstract
The Transcreener OAADPr SIRT FP Assay is a biochemical HTS assay for measuring the production of 2’-O-acetyl-ADP-ribose (OAADPr) by sirtuins (SIRT), NAD-dependent deacetylases. 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) assay. As an example, the Transcreener OAADPr SIRT FP assay can be used to detect the activity of human SIRT1 and SIRT2 using acetylated peptides as substrates.
Image Attribution
Figure 1. Schematic Overview of the Transcreener OAADPr SIRT FP Assay.
Figure 2. Simple Mix-and-Read Format.
Figure 3. Enzyme Titration Curve.
Figure 4. AMP Standard Curves. Example concentrations for a standard curve corresponding to 50 μM NAD^+^ and sample standard curves for 0.1 μM, 1 μM, 10 μM, 100 μM, 500 μM, and 1,000 μM NAD^+^ used in a 10 μL SIRT Enzyme Reaction.
Guidelines
IMPORTANT: Antibody centrifugation is required to remove aggregates that can disrupt data quality. Antibodies should be centrifuged at 10,000 x g for 10 minutes before use. Following centrifugation, pipette the solution needed from the top of the aliquot to ensure precipitate is not present in the detection reagents.
Storage
The CE should be stored at -80°C; other reagents can be stored at -20°C. Though we have confirmed that the CE is stable and maintains 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 the date of receipt.
Materials
AMP2/GMP2 Antibody: 1.26 mg/mL solution in PBS with 10% glycerol*
AMP2/GMP2 Alexa Fluor 633 Tracer: 800 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-35
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
Stop 26 Detect Buffer B, 10X: 200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35
NAD+, 5 mM: 5 mM in water
AMP, 5 mM: 5 mM in water
Enzyme Assay Buffer G, 10X: 500 mM Tris (pH 7.5), 50 mM MgCl_2, and 0.1% Triton X-100
DTT, 1 M: 1 M in water
MnCl_2, 1 M: 1 M in water
*NOTE: The exact concentration may vary from batch to batch. Please refer to the Certificate of Analysis for an accurate concentration.
Ultrapure Nuclease Free Water: Use nuclease free water such as: Invitrogen Part # AM9930
Enzyme: The Transcreener OAADPr SIRT FP Assay is designed for use with purified SIRT enzymes, which are available from BellBrook as stand-alone products (Part# 2340, 2341, 2342, 2343) and in SIRT1 and SIRT2 EnzoLution Assay Kits (Part# 3048, 3049).
Acetylated Peptide: The assay has been validated using H3K9Ac (1-21), an acetylated peptide derived from the N-terminus of Histone H3. H3K9Ac (1-21) is supplied in SIRT1 and SIRT2 EnzoLution Assay Kits (Part# 3048 and 3049).
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.
Liquid Handling Devices: Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates.
Assay Plates: It is important to use assay plates that are entirely black with a nonbinding surface. We recommend Corning® 384-well plates (Cat.# 4514). The suggested plate has a square well top that enables efficient plate washing and a round bottom that allows good Z' factors. It has a recommended working volume of 15–20 μL.
Troubleshooting
Safety warnings
NOTE: The AMP2/GMP2 Antibody concentration may vary by lot, so the volume required for a 10 μg/mL concentration should be adjusted accordingly.
NOTE: Pipetting small sample volumes accurately requires the correct equipment and proper technique. An extra dilution step may be required to ensure accuracy.
Before start
Note: Read the entire protocol and note any reagents or equipment needed (see Section 2.2).
Introduction
The Transcreener OAADPr SIRT FP Assay is a biochemical HTS assay for measuring the production of 2’-O-acetyl-ADP-ribose (OAADPr) by sirtuins (SIRT), NAD-dependent deacetylases. 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) assay. As an example, the Transcreener OAADPr SIRT FP assay can be used to detect the activity of human SIRT1 and SIRT2 using acetylated peptides as substrates.
Key Specifications:
- Single-addition, mix-and-read format
- Signal stability > 24 hours
- Excellent data quality (Z’ ≥ 0.7)
- Minimum interference from fluorescent compounds and light scattering with far-red tracer
Key Applications:
- Screening for enzyme inhibitors/activators
- Generating dose-response curves and IC50 values for inhibitors
- Kinetic and mechanistic analyses
Figure 1. Schematic Overview of the Transcreener OAADPr SIRT FP Assay. 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 reaction is quenched by EDTA, and AMP displaces an Alexa Fluor® 633 Tracer from the AMP2/GMP2 Antibody, resulting in decreased fluorescence polarization.
Product Specifications
| Product | Quantity | Part# | |
| Transcreener OAADPr SIRT FP Assay | 1,000 assays* | 3046-1K | |
| 10,000 assays* | 3046-10K |
*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.
IMPORTANT: Antibody centrifugation is required to remove aggregates that can disrupt data quality. Antibodies should be centrifuged at 10,000 x g for 10 minutes before use. Following centrifugation, pipette the solution needed from the top of the aliquot to ensure precipitate is not present in the detection reagents.
Storage
The CE should be stored at -80°C; other reagents can be stored at –20°C. Though we have confirmed that the CE is stable and maintains 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 the date of receipt.
Materials Provided
| Component | Composition | Notes | |
| AMP2/GMP2 Antibody | 1.26 mg/mL solution in PBS with 10% glycerol* | Sufficient antibody is included in the kit to complete 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 | The final tracer concentration in the 20 µL Complete Assay is 4 nM. Sufficient tracer is included in the kit to complete 1,000 assays (Part# 3046-1K) or 10,000 assays (Part# 3046-10K). | |
| 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 | Sufficient CE for 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. | |
| NAD+, 5 mM | 5 mM in water | Sufficient NAD+ is included in the kit to complete 1,000 assays (Part# 3046-1K) or 10,000 assays (Part# 3046-10K). | |
| AMP, 5 mM | 5 mM in water | The AMP can be used to create a standard curve for conversion of mP values to amount AMP formed, which is equimolar to OAADPr formation. | |
| 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. |
*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 | |
| Enzyme | The Transcreener OAADPr SIRT FP Assay is designed for use with purified SIRT enzymes, which are available from BellBrook as stand-alone products (Part# 2340, 2341, 2342, 2343) and in SIRT1 and SIRT2 Enzolution Assay kits (Part# 3048, 3049). It is not recommended for use with cell lysates or impure enzyme preparations as contaminating enzymes, such as phosphatases or nucleotidases, can interfere with the assay and reduce the assay window. | |
| Acetylated Peptide | The assay has been validated using H3K9Ac (1-21), an acetylated peptide derived from the N-terminus of Histone H3. H3K9Ac (1-21) is supplied in SIRT1 and SIRT2 Enzolution Assay kits (Part# 3048 and 3049). | |
| 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. | |
| Assay Plates | It is important to use assay plates that are entirely black with a nonbinding surface. We recommend Corning® 384-well plates (Cat.# 4514). The suggested plate has a square well top that enables easier robotic pipetting and a round bottom that allows good Z’ factors. It has a recommended working volume of 15–20 µL. |
Before You Begin
Read the entire protocol and note any reagents or equipment needed (see Section 2.2).
Plate Reader Configuration
Filters and Settings
To confirm that your plate reader is capable of measuring FP (not simply fluorescence intensity) of the AMP2/GMP2 Alexa Fluor 633 Tracer, please see our Instrument Compatibility page, which has links for instrument-specific application notes. Please contact BellBrook Labs Technical Support if you have questions about settings and filter sets for a specific instrument.
Define the Maximum Signal Window for the Instrument
Measuring high (tracer + antibody) and low (free tracer) polarization values will define the maximum
assay window for your specific instrument. Prepare High and Low FP controls in quantities sufficient to
perform at least 6 replicates for each condition, as described below, and measure the polarization
values. Subtract the Low FP Control readings from the corresponding High FP Control readings; the
difference should be >150 mP.
High FP Control:
| Component | As Provided | Final Concentration in 20 µL Complete Assay | Example: 25 Assays | Your Numbers | |
| AMP2/GMP2 Antibody | 1.26 mg/mL* | 10 µg/mL | 4.76 µL** | ||
| AMP2/GMP2 Alexa Fluor 633 Tracer | 800 nM | 4 nM | 3 µL | ||
| Stop & Detect Buffer B | 10X | 0.5X | 30 µL | ||
| Water | 562.24 µL | ||||
| Total | 600 µL |
*NOTE: The AMP2/GMP2 Antibody concentration may vary by lot, so the volume required for a 10
μg/mL concentration should be adjusted accordingly.
**NOTE: Pipetting small sample volumes accurately requires the correct equipment and proper
technique. An extra dilution step may be required to ensure accuracy.
Low FP Control:
| Component | As Provided | Final Concentration in 20 µL Complete Assay | Example: 25 Assays | Your Numbers | |
| AMP2/GMP2 Alexa Fluor 633 Tracer | 800 nM | 4 nM | 3 µL | ||
| Stop & Detect Buffer B | 10X | 0.5X | 30 µL | ||
| Water | 567 µL | ||||
| Total | 600 µL |
Calculate the Optimal AMP2/GMP2 Antibody Concentration
NOTE: This step is not required if you are using one of BellBrook’s Enzolution SIRT Assay Systems.
The sensitivity and dynamic range of the Transcreener OAADPr SIRT FP Assay are determined by the concentration of the AMP2/GMP2 antibody. A lower antibody concentration increases sensitivity but may reduce the dynamic range, while a higher antibody concentration results in lower sensitivity but may extend the dynamic range.
The required AMP2/GMP2 antibody concentration in the 1X AMP Detection Mix depends on the amount of NAD+ used in the assay and can be calculated using the following equation:
[Ab] (μg/ml) = 0.3274 x [NAD+] (μM) + 4.24
For example, if 50 μM NAD+ is used in a 10 μL Enzyme Reaction, the optimal AMP2/GMP2 antibody concentration for 10 μL of 1X AMP Detection Mix would be 20.6 μg/mL.
Protocol
Performing an Enzyme Assay
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.
The following assay protocol is for 384-well format, using a 10 μL Enzyme Reaction and 20 μL Complete Assay volume when the plates are read. The use of different format will require changes in reagent quantities (see Section 6.1). 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 SIRT enzyme to 2X the desired concentration in Complete Assay Buffer.
c. Prepare Substrate/CE Mix by combining 2X CE, 2X the desired concentration of NAD+ and peptide in Complete Assay Buffer.
2. Run the SIRT Enzyme Reaction
a. Add 5 μL of 2X SIRT 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 combining 1X Stop & Detect Buffer B, 8 nM AMP2/GMP2
Tracer, and an appropriate concentration of AMP2/GMP2 Antibody (see Section 3.2) in
Ultrapure Nuclease-Free Water.
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.
1x AMP Detection Mix based on 50 μM NAD+ in the 10 µL Enzyme Reaction:
1X AMP Detection Mix - Add 10 µL Per Well
| Component | As Provided | Detection Mix Concentration | Final Concentration in 20 µL Complete Assay | Final Volume in AMP Detection Mix (1X) | |
| AMP2/GMP2 Antibody | 1.26 mg/mL | 20 µg/mL | 10 µg/mL | 158.6 µL | |
| AMP2/GMP2 Alexa Fluor 633 Tracer | 800 nM | 8 nM | 4 nM | 100 µL | |
| Stop & Detect Buffer B | 10X | 1X | 0.5X | 1000 µL | |
| Water | 8,741.4 µL | ||||
| Total | 10,000 µL |
Note: Each time an assay is performed with a new batch of enzyme, an enzyme titration is required to determine the working concentration.
For inhibitor screening and profiling, we recommend using an enzyme concentration that produces 50–
80% of the maximal signal (EC50 to EC80) (see Figure 3) and an assay window of at least 100 mP.
Meeting these parameters will ensure a) a robust signal, resulting Z’ values >0.7 and good sensitivity
to inhibition and b) initial velocity conditions for the enzyme reaction, which corresponds to the linear
phase of the reaction (after conversion of mP values to AMP formed). 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. Enzyme Titration Curve. Example SIRT1 Enzyme titration. For screening and profiling
inhibitors, the ideal range of enzyme concentrations is between EC50 and EC80.
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.
Performing an AMP 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.
| Data Point | AMP (µM) | NAD+ (µM) | |
| 1 | 50 | 50 | |
| 10 | 0.098 | 50 | |
| 11 | 0.049 | 50 | |
| 12 | 0 | 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 |
Figure 4. AMP Standard Curves. Example concentrations for a standard curve corresponding to 50 μM NAD+ and sample standard curves for 0.1 μM, 1 μM, 10 μM, 100 μM, 500 μM, and 1,000 μM NAD+ used in a 10 μL SIRT Enzyme Reaction.
General Considerations
Reagent an Signal Stability
Signal Stability
The stability of the FP assay window at 10% substrate conversion was determined after the addition of the 1X AMP Detection Mix to the standard samples. The FP assay window at 10% substrate conversion remained constant (<10% Change) for at least 24 hours at room temperature (20–25°C). If you plan to read FP on the following day, seal the plates to prevent evaporation.
AMP Detection Mix Stability
The AMP Detection Mix is stable for at least 16 hours at room temperature (20–25°C). If you prepare the AMP Detection Mix more than 30 minutes before addition, store it on ice or at 4°C until needed to help decrease the equilibration time.
Enzyme Assay Controls
The enzyme reaction controls define the limits of the enzyme assay.
| Component | Notes | |
| No Inhibitor Control | This is a complete Enzyme Reaction with all detection components. It provides the maximum signal (minimal FP value) for an uninhibited Enzyme Reaction. | |
| No Enzyme Control | This contains all Enzyme Reaction components in the absence of the target enzyme. It provides the minimal signal (maximal FP value), mimicking 100% Inhibition. | |
| Minus-Substrate Control | This is an alternative to a No Enzyme Control; it may be more appropriate for enzymes that produce some background signal that is not substrate-dependent. |
Frequently Asked Questions
| Question | Possible Solutions | |
| Low Selectivity | Suboptimal antibody concentration
| |
| No change in FP observed | Low antibody/tracer activity or Δ mP signal.
| |
| Is a standard curve required? | No, it is not required to run a standard curve. We recommend running the AMP standard curve if you want to convert raw mP values to product formed. While designing a standard curve, make sure that most of the points are within the area of interest (initial velocity conditions). We do not recommend using a standard curve from previous experiments, rather generate a new curve with each experiment to achieve the most accurate result. | |
| Can this assay be used with cell lysates? | The assay will only work with purified recombinant protein. The presence of nucleases in the lysates prohibits the use of Transcreener assays with lysates. | |
| High background signal or change in signal after incubation with detection mixture. | Interference from impurities
|
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