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Transcreener pADPr PARP Assay measures poly(ADP-ribose) (pADPr) produced in poly(ADP-ribose) polymerase (PARP) reactions. pADPr measurements allow researchers to effectively determine the enzyme’s activity. The assay provides a powerful tool to screen compound libraries for PARP modulators to help find new therapies for disease.
The PARP activity assay kit comes complete with the detection reagents required to measure activity. PARP enzyme is not included in the inhibitor screening assay kit. Recombinant PARP1 and PARP2 enzymes, as well as Enzolution PARP1 and PARP2 Assay Systems, are available for separate purchase through BellBrook Labs.
This PARP assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.
The Transcreener pADPr PARP FP Assay is a biochemical HTS assay for measuring the production of poly(ADP-ribose) (pADPr) in poly(ADP-ribose) polymerase (PARP) reactions.
The assay relies on pADPr Coupling Enzymes (CE) to convert pADPr into AMP, which is then detected using a far-red, competitive fluorescence polarization (FP) or TR-FRET assay. Detection is powered by a highly selective pADPr antibody, enabling sensitive measurement of ADP-ribose production.
As an example, the assay can be used to detect the activity of human PARP1, which uses NAD+ to form pADPr on itself, histones, and DNA repair proteins. The Transcreener pADPr PARP Assay is designed specifically for high throughput screening (HTS), with a single-addition, mix-and-read format.
It is easy to integrate into automated HTS workflows, with outstanding reagent stability (deck stability > 16 hours, signal stability > 24 hours), robust detection of ADPR monomers released from pADPr over a range of 10 nM to 100 μM, and compatibility with commonly used multimode plate readers. Data quality is excellent (Z’ ≥ 0.7), and the assay uses a far-red tracer to minimize interference from fluorescent compounds and light scattering.
This video highlights a high-throughput, mix-and-read biochemical assay for quantitative PARP activity and inhibitor screening. Using enzymatic conversion of poly(ADP-ribose) to AMP with Transcreener® detection, the assay delivers exceptional sensitivity, automation compatibility, and accurate IC₅₀ determination for PARP drug discovery.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The PARP assay is compatible with 96, 384, and 1536-well formats, making it ideal for scalable screening campaigns.
PARP1 enzyme titration using the Enzolution PARP1 Assay System. The assay demonstrates linearity when raw data is converted to AMP using a standard curve.
PARP2 enzyme titration using the Enzolution PARP2 Assay System. The assay demonstrates linearity when raw data is converted to AMP using a standard curve.
The Transcreener pADPr Assay is designed for screening compound libraries in a high throughput format. Follow up SAR can also be performed using the assay to determine inhibitor potency with ease.
Example dose-response curves with known PARP1 inhibitors. Data was generated using the Transcreener pADPr PARP FP Assay, with the Enzolution PARP1 Assay System. The following conditions were used. 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2. 0.6 nM full-length human PARP1, 50 µM NAD+, 0.25 mg/mL Sheared Salmon Sperm DNA, with 1X pADPr coupling enzyme mix. The enzyme was incubated with the test compound for 30 minutes before the addition of 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.
Active, full-length human PARP1 (AA 1-1014) protein expressed and purified from Baculovirus infected insect cells with an N-Terminal FLAG tag. The enzyme has been thoroughly validated with the Transcreener pADPr Assay Kit. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is >80% in a composition of 25 mM HEPES, 300 mM NaCl, 0.04% Triton X-100, 0.5 mM TCEP, and 10% Glycerol (pH 7.5). Currently used with the Transcreener assay at a concentration of between 0.6 nM to 1 nM in a 10 μL reaction. Please adjust the enzyme concentration based on your requirements.
Active, full-length human PARP2 (AA 1-583) protein expressed and purified from Baculovirus-infected insect cells with an N-terminal His tag. The enzyme has been thoroughly validated with the Transcreener pADPr Assay Kit. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is >90% in a composition of 50 mM TRIS, 500 mM NaCl, and 5% Glycerol (pH 8.0). Currently used with the Transcreener assay at a concentration of 3 nM in a 10 μL reaction. Please adjust the enzyme concentration based on your requirements.
There is extensive crosstalk between the DNA damage response (DDR) pathways and innate immune pathways. Both the individual pathways and the interconnections between them are a focus for exciting new small molecule drug therapeutics that target cancers and debilitating autoimmune disorders. The Transcreener HTS Assay platform accelerates these efforts by providing a robust and easy-to-use biochemical assay to measure activity of key enzymes in the innate immune and DDR pathway.
In this guide, we provide an overview of the DDR and innate immune pathway, and describe Transcreener Assays and Assay Systems for key therapeutic targets. The enzyme targets discussed include:
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Enzolution® PARP1 Assay System
