TR-FRET PARP2 Trapping Assay Kit

Background

PARP2 (Poly (ADP-ribose) polymerase 2) is a member of the PARP family and plays a crucial role in DNA repair, particularly in the repair of single-strand breaks (SSBs) in DNA. It binds to DNA at the site of damage, becomes catalytically activated, and uses NAD⁺ as a substrate to add poly (ADP-ribose) (PAR) chains to itself and other proteins—a process called PARylation that results in the recruitment of other DNA repair proteins to the damaged site. Because of the high negative charge of PAR polymers, extensive autoPARylation of PARP2 leads to the dissociation of PARP2 from DNA, which is required for DNA repair completion. PARP2 is often overexpressed in various cancers, including breast, ovarian, prostate, lung, and glioblastoma. This overexpression is thought to support tumor cell survival. Some PARP inhibitors not only block the catalytic activity of PARP2 but also trap PARP2 on DNA at sites of damage, preventing its release. This creates a toxic DNA-protein complex that interferes with DNA replication and repair, leading to cell death, particularly in cancer cells deficient in homologous recombination repair (e.g., BRCA1/2-mutant cells).

Assay Principle

The TR-FRET PARP2 Trapping Assay Kit is designed to detect the poly-ADP-ribosylation activity of PARP2 and the status of PARP2 trapping on DNA. The DNA substrate in the kit is labeled with a fluorophore (acceptor). A Terbium (Tb)-labeled anti-Tag2 antibody that binds to Tag2-Kras serves as the fluorescence donor. Activation of Tb results in fluorescence resonance energy transfer (FRET) if PARP2 binds to the fluorescence-labeled DNA, since the binding brings the fluorescence donor into close proximity with the fluorophore acceptor. Thus, the binding status can be quantitatively measured by calculating the ratio of the emission fluorescence intensities of the acceptor (665 nm) and the donor (620 nm). In the presence of NAD⁺, auto-PARylation of PARP2 leads to its dissociation from DNA, resulting in a decrease in the FRET signal. Inhibition of auto-PARylation activity traps PARP2 on the DNA, and the FRET signal remains high.

Application

High throughput screening of compounds that inhibit the auto-PARylation activity of PARP2 for drug discovery.

Instrument Required

A HTRF® certified microplate reader capable of measuring Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) is required.

Kit Components

Materials Not Supplied

• Microplate reader, HTRF® certified microplate reader
• Adjustable micro-pipettor
• Sterile Tips

Assay Protocol

1. Step 1. Prepare PARP2 solution Thaw PARP2 protein on ice. Upon first thaw, briefly spin tube to recover the full contents at the bottom of the tube. Make aliquots of the enzyme for single use. Store remaining undiluted protein at -80°C. Note: PARP2 protein is sensitive to freeze/thaw cycles. Limit number freeze-thaw cycles for best results. Do not re-use the diluted protein. Dilute the PARP2 protein 500-fold (1 µL PARP2 + 499 µL assay buffer). Add 4 µl of diluted protein solution to each of positive control wells and inhibitor test wells. Add 4 µl of assay buffer to each of negative control wells.
2. Step 2. Add inhibitor Add 2 µl of diluted compound solution to each inhibitor test well. Add 2 µl of inhibitor solvent solution to each of negative and positive control well.
3. Step 3. Prepare the DNA substrate solution Dilute the fluorescence-labeled DNA 20-fold (1 µL DNA + 19 µL assay buffer). Add 4 µl of the diluted DNA solution to each well.
4. Step 4. Prepare NAD+ solution Dilute the NAD+ 25-fold (1 µL NAD+ + 24 µL assay buffer). Add 5 µl of diluted NAD+ solution to each of positive control and compound test wells.
5. Step 5. Prepare dye solution Dilute Terbium-labeled anti-Tag2 antibody 1:100. For example: 1 µl of Terbium-labeled anti-Tag2 antibody + 99 µl assay buffer. Add 5 µl of this dye mixture to each well.
6. Step 6. Incubate the reaction at room temperature for 30 minutes.
7. Step 7. Measure fluorescent intensity HTRF compatible microplate reader is needed to measure fluorescent intensity of the samples. Fluorescent intensity should be measured twice:
8. Step 8. Excitation wavelength at 340 nm and emission at 620 nm.
9. Step 9. Excitation wavelength at 340 nm and emission at 665 nm.

Data Analysis

Calculate sample HTRF signal of each well. Calculate percentage activity In the absence of the compound (positive control), the sample signal (P) is defined as 100% activity. In the absence of enzyme (negative control), the sample signal (N) is defined as 0% activity. The percent activity in the presence of each compound is calculated according to the following equation: % activity = (S-N)/(P-N) X100, where S= the sample signal in the presence of the compound.

Assay Validation

Biological Pathway / Process

DNA Damage Response (DDR)

Therapeutic / Disease Area

Oncology (breast; ovarian; prostate)