Jan 30, 2026

Public workspaceEnzolution™ PARP2 Assay System Technical Manual

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvod45dg4o/v1
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvod45dg4o/v1
Protocol Citation: info info 2026. Enzolution™ PARP2 Assay System Technical Manual. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvod45dg4o/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: November 06, 2025
Last Modified: January 30, 2026
Protocol Integer ID: 231616
Keywords: parp2 assay system, parp2 assay system tech manual, parp2 assay system technical manual, enzolution parp2 assay system, parp2 assay system technical manual the enzolution, parp2 assay system, parp2 assay system technical manual, transcreener padpr parp assay kit, enzymatic activity of parp2, parp2 inhibitor, parp2, polymerase, full length human parp2, inhibitor dose response measurement, assay, coupling enzyme, target protein, other protein substrate, chains on target protein, addition of other protein substrate, enzymatic activity, ribosylation reaction, donor substrate, high throughput screening, padpr
Abstract
The Enzolution™ PARP2 Assay System is intended for use with the Transcreener® pADPr PARP Assay Kits (Parts #3043 and #3044) to measure the enzymatic activity of PARP2 (poly(ADP-ribose) polymerase 2). Upon binding to DNA, PARP2 forms poly(ADP-ribose) (pADPr) chains on target proteins, including itself, using NAD+ as a donor substrate (Figure 1). The assay relies on Coupling Enzymes (CE) to convert pADPr into AMP, which is detected using a far-red, 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 PARP2 Assay System provides all reagents required to screen and profile PARP2 inhibitors when used with the Transcreener pADPr PARP Assay Kits, including purified, full length human PARP2 and sheared salmon sperm DNA (“sssDNA” in Figure 1). Since PARP2 can add pADPr to itself, this assay was optimized to detect the auto-ADP-ribosylation reaction and does not require the addition of other protein substrates. 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™ PARP2 Assay System is intended for use with the Transcreener® pADPr PARP Assay Kits (Parts #3043 and #3044) to measure the enzymatic activity of PARP2 (poly(ADP-ribose) polymerase 2). Upon binding to DNA, PARP2 forms poly(ADP-ribose) (pADPr) chains on target proteins, including itself, using NAD+ as a donor substrate (Figure 1). The assay relies on Coupling Enzymes (CE) to convert pADPr into AMP, which is detected using a far-red, 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 PARP2 Assay System provides all reagents required to screen and profile PARP2 inhibitors when used with the Transcreener pADPr PARP Assay Kits, including purified, full length human PARP2 and sheared salmon sperm DNA (“sssDNA” in Figure 1). Since PARP2 can add pADPr to itself, this assay was optimized to detect the auto-ADP-ribosylation reaction and does not require the addition of other protein substrates. 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 PARP2 inhibitors (or activators)
  • Generating dose response curves and IC50 values for PARP2 inhibitors
  • Kinetic and mechanistic analyses

Figure 1. Schematic Overview of the Enzolution PARP2 Assay System with the Transcreener pADPr PARP Assays. pADPr produced by PARP2 is completely converted to AMP in real time by the pADPr Coupling Enzymes. 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

ProductQuantityPart #
Enzolution PARP2 Assay System1,000 assays*3050-1K
10,000 assays*3050-10K
*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

ComponentCompositionNotes
PARP2 Enzyme0.5 mg/mL (7.46 μM)* in 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerolFull-length protein (amino acids 1-583), N-Terminal His tag, 67 kDa. The exact concentration may vary from batch to batch. Please refer to the Certificate of Analysis for and accurate concentration.
Sheared Salmon Sperm DNA10 mg/mL in waterSalmon Sperm DNA stored in Nuclease Free Water; to be used at 0.25 mg/mL in the PARP2 reaction.
Enzyme Assay Buffer H, 10X500 mM Tris (pH 8.0), 50 mM MgCl2, 1 M NaCl, and 0.1% TritonEnzyme Assay Buffer H, along with DTT, has been optimized to support PARP2 activity as well as CE activity. Changes to the assay buffer may affect PARP2 enzyme activity and/or conversion of pADPr to AMP.
DTT, 1 M1 M in waterAdd to 1x Enzyme Assay Buffer H to a final concentration of 1 mM.
382-Well Low Volume Assay PlatesCorning# 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.

Materials Required but Not Provided

ComponentNotes
Ultrapure Nuclease Free WaterSome 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 ReaderList of compatible plate readers and settings.
Liquid Handling DevicesUse liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates.
Laboratory IncubatorAn incubator model that is capable of maintaining temperature stability at 30°C is required.

Transcreener® pADPr PARP TR-FRET Assay - SOLD SEPARATELY
ComponentCompositionNotes
AMP2/GMP2 Antibody-Tb800 nM solution in 25 mM HEPES buffered saline1,000 assays (Part # 3044-1K) or 10,000 assays (Part # 3044-10K)
AMP2/GMP2 2 Hilyte 647 Tracer10 μM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-351,000 assays (Part # 3044-1K) or 10,000 assays (Part # 3044-10K)
Stop & Detect Buffer C, 10X500 mM HEPES, 0.2% Brij, and 200 mM EDTA at a final pH 7.5The Stop & Detect Buffer C components quench the CE reaction by chelating metals required for activity.
pADPr Coupling Enzymes (CE)400X pADPr Coupling EnzymepADPr Coupling Enzymes are present in excess to ensure pADPr is completely converted to AMP.
NAD+5 mM in waterThe use of NAD from different sources may require reoptimize the assay’s dynamic range.
AMP5 mM in waterUsed for the AMP standard curve.

Transcreener® pADPr PARP FP Assay - SOLD SEPARATELY
ComponentCompositionNotes
AMP2/GMP2 Antibody1.26 mg/mL solution in PBS with 10% glycerol1,000 assays (Part # 3043-1K) or 10,000 assays (Part # 3043-10K).
AMP2/GMP2 Alexa Fluor 633 Tracer800 nM solution in 2 mM HEPES (pH 7.5) containing 0.01% Brij-351,000 assays (Part # 3043-1K) or 10,000 assays (Part # 3043-10K).
Stop & Detect Buffer B, 10X200 mM HEPES (pH 7.5), 400 mM EDTA, and 0.2% Brij-35The Stop & Detect Buffer B components quench the CE reaction by chelating metals required for activity.
pADPr Coupling Enzyme (CE)400X pADPr Coupling EnzymepADPr Coupling Enzyme is present in excess to ensure pADPr is completely converted to AMP.
NAD+5 mM in waterThe use of NAD from different sources may require reoptimize the assay’s dynamic range.
AMP5 mM in waterUsed for the AMP standard curve.

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® pADPr PARP Assays. Full List of Compatible Plate Reader Settings
  • Please read and understand the Transcreener® pADPr PARP Assay Technical Manuals prior to use with this kit.
Protocol
The methods described below are for endpoint detection of pADPr formation by PARP2 under initial velocity conditions (≤ 20% conversion of NAD+ to pADPr) with a sub-Km concentration of NAD+ (100μM) and a saturating concentration of sheared salmon sperm DNA (0.25 mg/mL). These conditions will ensure sensitive detection of inhibitors that compete with NAD+. Significant changes in the NAD+ concentration may require adjustment of the dynamic range, as described in the Transcreener pADPr PARP Assay Tech Manuals.

Figure 2. Simple Mix-and-Read Format. The PARP2 Enzyme Reaction is run in the presence of CE, so that pADPr 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.

Performing an Enzyme Assay
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 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 H, and 1 mM DTT in Ultrapure Nuclease-Free Water. b. Dilute PARP2 enzyme to 2X the desired concentration* in Complete Assay Buffer. c. Prepare Substrate/CE Mix by combining 2X CE, 200 µM NAD+, 0.5 mg/mL sheared salmon sperm DNA in Complete Assay Buffer.

10 µL Enzyme Reaction Components
ComponentStockWorking StockFinal Concentration in 10 µL
Complete Assay Buffer10X Enzyme Assay Buffer H, 1 M DTT1X Enzyme Assay Buffer H, 1 mM DTT in Nuclease Free Water50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 100 mM NaCl, 0.01% Triton X-100, 1 mM DTT
PARP2 Enzyme0.5 mg/mL (7.46 μM)2X in Complete Assay Buffer3 nM – 4 nM (see Section 4.1)
Substrate/CE Mix5 mM NAD+, 10 mg/mL sheared salmon sperm DNA, 400X CE200 µM NAD+, 0.5 mg/mL sheared salmon sperm DNA, 2X CE in Complete Assay Buffer100 µM NAD+, 0.25 mg/mL salmon sheared sperm DNA, 1X CE
Table 1. PARP2 Enzyme Reaction Components.

2. Run the PARP2 Enzyme Reaction a. Add 5 μL of 2x PARP2 enzyme to each well. b. Add 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.

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.
*Using the EC80 concentration suggested in the PARP2 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) mP values from A) are 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 Curves of probe inhibitors (see Section 4.2). (d) Z’ Measurement (n=12) (see Section 4.4).

Performing Single Compound Screening and Dose-Response Assays
For Single Compound Screening and Dose-Response Assays, follow the protocol listed in Section 4.1. Add PARP2 enzyme 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 RT to allow equilibration of the E-I complex.
NOTE: Final concentration of test compounds should be based on the volume of the Enzyme Reaction (10 μL).
Setting Up a Standard Curve
Use of a standard curve for conversion of raw FP or TR-FRET data 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 PARP2 enzyme replaced by a titration of AMP pADPr concentrations. The Substrate/CE Mix remains the same as in the PARP2 Enzyme Reaction to account for background generated by NAD+ degradation.
Typically, an 8- to 12-point standard curve is used, with the starting concentration of AMP matching the NAD+ concentration in the PARP2 Enzyme Reaction (see Figure 4). 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 200 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 200 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 as described in Section 4.

Data Point AMP (µM) NAD+ (µM)
1100100
250100
325100
412.5100
56.25100
63.125100
71.56100
80.78100
90.39100
100.195100
110.0976100
120100

Figure 4. AMP Standard Curves. Example concentration and data for a standard curve corresponding to 100 μM NAD+ in the PARP2 Enzyme Reaction. Note that NAD+ would decrease proportionally in a PARP reaction, but it can be held constant to simplify the standard curve protocol.

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.

ComponentTotal VolumeEnzyme Reaction Volume 1X AMP Detection Mix Volume
96 Well Low Volume Plate50 µL25 µL25 µL
384 Well Low Volume Plate20 µL10 µL10 µL
1536 Well Low Volume Plate8 µL4 µL4 µL

Links to Applicable Application Notes
Contact Information
Email: [email protected] Phone: 608.443.2400 Toll-Free: 866.313.7881 FAX: 608.441.2967