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An Enzyme-Linked Immunosorbent Assay to Quantify Poly (ADP-Ribose) Level In Vivo

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Poly(ADP-Ribose) Polymerase

Abstract

PolyADP-ribosylation is a posttranslational modification of proteins that results from enzymatic synthesis of poly(ADP-ribose) with NAD+ as the substrate. A unique characteristic of polyADP-ribosylation is that the poly(ADP-ribose) chain can have 200 or more ADP-ribose residues in branched patterns, and the presence and variety of these chains can have substantive effects on protein function. To understand how polyADP-ribosylation affects biological processes, it is important to know the physiological level of poly(ADP-ribose) in cells. Under normal cell physiological conditions and in the absence of any exogenous DNA damaging agents, we found that the concentration of poly(ADP-ribose) in HeLa cells is approximately 0.04 pmol (25 pg)/106 cells, as measured with a double-antibody sandwich, enzyme-linked immunosorbent assay protocol that avoids artificial activation of PARP1 during cell lysis. Notably, this system demonstrated that the poly(ADP-ribose) level peaks in S phase and that the average cellular turnover of a single poly(ADP-ribose) is less than 40 s.

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Acknowledgments

We thank Drs. Susumu Ikegami and Takahiro Fujii of the Nagahama Institute of Bio-Science and Technology and the late Dr. Shin Ogata of Mie University, for valuable comments and support. The technical assistance of Mr. Toru Kida, Mr. Masaki Tsukada, Mr. Teruaki Sato, Ms. Narumi Ohta, Ms. Mie Tsuchida, and Ms. Miho Hosoi is greatly acknowledged. This work was supported partly by the Grants-in-Aid for Scientific Research (23590350) from the Japan Society for the Promotion of Science to M.M. and partly by the Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology-Japan (JP21H03547) to T.S. J.M. was supported by the Intramural Research Program, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH), USA. This work was performed partly in the Cooperative Research Project Program of the Medical Institute of Bioregulation, Kyushu University, Japan.

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Correspondence to Masanao Miwa .

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Ida, C. et al. (2023). An Enzyme-Linked Immunosorbent Assay to Quantify Poly (ADP-Ribose) Level In Vivo. In: Tulin, A.V. (eds) Poly(ADP-Ribose) Polymerase. Methods in Molecular Biology, vol 2609. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2891-1_6

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  • DOI: https://doi.org/10.1007/978-1-0716-2891-1_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2890-4

  • Online ISBN: 978-1-0716-2891-1

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