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Application: Study enzyme kinetics and screen small molecule inhibitors for drug discovery and high throughput screening (HTS) applications.
Background: The (Poly-ADP-ribose) polymerase (PARP) family of proteins are involved in DNA repair, genomic stability, and programmed cell death. They catalyze the NAD-dependent addition of poly(ADP-ribose) to histones. PARP3 is critical in mitotic progression by stabilizing the mitotic spindle via regulation of NuMA and Tankyrase 1. PARP3 overexpression is found in patients with breast cancer and is associated with poor prognosis. PARP inhibition can be highly advantageous in tumors that involve BRCA1/2 (breast cancer type 1/2 susceptibility protein) mutations, as it leads to accumulation of DNA single strand breaks (SSBs) that are not repaired and cell death. PARP inhibitors work through "synthetic lethality", where cell death is due to impaired function of two or more genes, but a defect in a single gene is not lethal. PARP3 inhibitors are thus therapeutical relevant in the treatment of metastatic breast cancer.
Contraindications: The PARP3 Chemiluminescent Assay Kit is compatible with up to 1% final DMSO concentration. We recommend preparing the inhibitor in no higher than 10% DMSO solution in buffer and using 5 µl or 2.5 µl per well.
Description: The PARP3 Chemiluminescent Assay Kit is designed to measure PARP3 activity for screening and profiling applications. The PARP3 assay kit 96-well format contains enough purified PARP3 enzyme, histone mixture, activated DNA template, PARP assay buffer and necessary reagents for 100 or 400 enzyme reactions. The key to the PARP3 Chemiluminescent Assay Kit is the biotinylated NAD+ and involves 3 reactions. First, histone proteins are coated on a plate. Next, a biotinylated NAD+ mix (termed PARP Substrate Mixture) is incubated with the enzyme and an activated DNA template. Finally, streptavidin-HRP is added, followed by ELISA ECL substrate to produce a chemiluminescence signal that can be measured using a chemiluminescence plate reader.*NOTE: As of April 2022, this protocol has been re-optimized for performance. Previous versions of this kit are available upon request.
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Supplied As: The PARP3 assay kit comes in a convenient 96-well format, with purified PARP3 enzyme, histone mixture, activated DNA, and PARP assay buffer for 100 enzyme reactions.
Uniprot: Q9Y6F1
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
References: Brown, J.A., Marala, R.B., J. Pharmacol. Toxicol. Methods 2002;47:137-41.
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