title: An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells.
abstract: PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells.
authors.affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan; Department of Applied Life Studies, College of Nagoya Women's University, Nagoya-shi, Aichi 467-8610, Japan.
authors.forename: Chieri
authors.initials: C
authors.lastname: Ida
authors.affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Yamashita
authors.affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Tsukada
authors.affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan.
authors.forename: Teruaki
authors.initials: T
authors.lastname: Sato
authors.affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Eguchi
authors.affiliation: Department of Microbiology, Kansai Medical University, Hirakata City, Osaka 573-1010, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Laboratory of Molecular and Cellular Biology, Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Ogata
authors.affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Fujii
authors.affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan.
authors.forename: Yoshisuke
authors.initials: Y
authors.lastname: Nishi
authors.affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Ikegami
authors.affiliation: Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Joel
authors.initials: J
authors.lastname: Moss
authors.affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan. Electronic address: m_miwa@nagahama-i-bio.ac.jp.
authors.forename: Masanao
authors.initials: M
authors.lastname: Miwa
mesh_terms: D000906:Antibodies / Q000276:immunology; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004249:DNA Damage; D003850:Deoxyribonuclease I / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006026:Glycoside Hydrolases / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D011064:Poly Adenosine Diphosphate Ribose / Q000032:analysis* / Q000276:immunology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015531:Radioimmunoprecipitation Assay; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D014238:Trichloroacetic Acid / Q000737:chemistry
chemical_list: D000906:Antibodies; D011064:Poly Adenosine Diphosphate Ribose; D014238:Trichloroacetic Acid; D011065:Poly(ADP-ribose) Polymerases; D003850:Deoxyribonuclease I; D015719:Single-Strand Specific DNA and RNA Endonucleases; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase
keywords: Antibody; ELISA; HeLa cells; Poly(ADP-ribose) glycohydrolase; Poly(ADP-ribose) polymerase