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Applications: Quantify total levels of PAR in cultured cells.Screen or determine the IC50 of PARP or PARG inhibitors in cultured cells.Validation of PARP or PARG inhibitors in a cellular model.
Background: PAR homeostasis is regulated by the family of PAR polymerases (PARPs) and PARG (Poly (ADP-ribose) glycohydrolase) in response to cellular stress conditions. ADP ribosylation, which is the addition of an ADP-ribose to a protein, is a reversible post-translational modification of proteins mostly involved in the DNA Damage Response (DDR) pathway. Poly-ADP-ribosylation (termed PARylation) is the addition of linear or branched chains of ADP-ribose. PARP and PARG activity are linked to cellular responses in inflammation, ischemia, stroke, and cancer. PARP inhibitors have been used in cancer treatment with success, leading to synthetic lethality when homologous recombination repair (HRR) is already defective. PARG is overexpressed in breast cancer and associated with tumor growth and survival. Decrease in PARG activity can potentiate the effect of current cancer therapies, such as chemotherapy and radiation, making PARG inhibition with selective inhibitors a promising approach in cancer and immunotherapy. Further studies of PARP and PARG will elucidate how the levels of PARylated proteins contribute to disease and can be modulated to provide therapeutic benefit.
Contraindications: This assay does not allow detection of mono-(ADP-ribose) (MAR) modifications on proteins.Avoid SDS or other strongly denaturing detergents at concentration > 0.1%.This assay is compatible with up to 2% v/v total detergent present and up to 1% DMSO.Avoid the use of sodium azide and reducing agents, such as dithiothreitol or β-mercaptoethanol at concentration >10 mM.
Description: LysA™ Universal PARylation Assay Kit is a sandwich ELISA-based kit designed to measure and quantify the amount of total poly ADP-ribosylation present in cell extracts. The kit comes in a convenient 96-well format and contains all the reagents needed, including a PAR standard that allows to establish a standard curve for quantitative measurements. It contains enough antibodies, blocking buffer, and detection reagents to measure PARylation levels in cell extracts, in a 96 well plate format. It also includes cell lysates as controls for assay performance.Note: The assay is adequate for Poly-ADP-ribosylation detection only. The assay is linear in the 100 pM to 20 nM PARylation range.An anti-PAR antibody is used to coat the 96-well plate. Lysates from cells are added to the coated wells, so PAR (PARylated proteins) present in the cell lysates are captured by the antibody. This is followed by an incubation with an anti-PAR primary antibody and a secondary HRP-labeled antibody. Addition of a chemiluminescent HRP substrate provides a luminescence signal, which directly correlates with the amount of PAR present in the cell extracts
Storage Stability: This assay kit will perform optimally for up to 6 months from date of receipt when the materials are stored as directed.
Warnings: Avoid freeze/thaw cycles
Biosafety Level: Not applicable (BSL-1)
References: Marques M., et al., 2019 Oncogene 38 (12): 2177-2191.
James D. I., et al., 2016 ACS Chem Biol 11 (11): 3179-3190.
Drown B. S., et al., 2018 Cell Chem Bio 25 (12): 1562-1570.
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