IDEAS home Printed from https://ideas.repec.org/a/gam/jchals/v9y2018i2p27-d155780.html
   My bibliography  Save this article

Measurement of Poly(ADP-ribose) Level with Enhanced Slot Blot Assay with Crosslinking

Author

Listed:
  • Yuko Kudo

    (Genome Stability Research Division, Lab of Collaborative Research, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
    Division of Materials Science & Chemical Engineering, Graduate School of Engineering, Yokohama National University, 79-5, Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan)

  • Yuka Sasaki

    (Division of Cell Signaling, Lab of Collaborative Research, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
    Department of Frontier Life Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan)

  • Takae Onodera

    (Division of Cell Signaling, Lab of Collaborative Research, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
    Department of Frontier Life Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan)

  • Jun Hashimoto

    (Department of Breast and Medical Oncology, National Cancer Center Hospital, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan)

  • Tadashige Nozaki

    (Department of Pharmacology, Faculty of Dentistry, Osaka Dental University, 8-1, Kuzuhahanazono-cho, Hirakata, Osaka 573-1121, Japan)

  • Kenji Tamura

    (Department of Breast and Medical Oncology, National Cancer Center Hospital, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, Japan)

  • Masatoshi Watanabe

    (Division of Materials Science & Chemical Engineering, Graduate School of Engineering, Yokohama National University, 79-5, Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan
    Current address: Oncologic Pathology Graduate School of Medicine, Mie University, 2-174 Edobashi, Tsu 514-8507, Japan)

  • Mitsuko Masutani

    (Genome Stability Research Division, Lab of Collaborative Research, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
    Division of Cell Signaling, Lab of Collaborative Research, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
    Department of Frontier Life Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan)

Abstract

Poly(ADP-ribose) (PAR) formation is catalyzed by poly(ADP-ribose) polymerase (PARP) family proteins in nuclei as well as in cytosols. The anti-PAR antibodies that specifically detect PAR are useful for the quantitative measurement of PAR in cells, in tissue, and in the body. In clinical trials of PARP inhibitors, a pharmacodynamic (PD) assay for the measurement of PARP activity inhibition in peripheral blood mononuclear cells (PBMCs) with dot-blot assay or an ELISA assay using anti-PAR antibodies have been used. In these assays, ex vivo PARP activity and its inhibition assay have been used. For a PD assay to assess the efficacy of the treatment, the measurement of PARP activity inhibition in tumor tissues/cells has been recommended. A dot or slot blot assay may also be suitable for the measurement of such crude tissue samples. Here, we investigate the optimum conditions for a dot/slot blot assay of an ex vivo PARP activity assay by utilizing physical and chemical crosslinking methods. Using 10H monoclonal antibody to PAR, we show that use of a nylon membrane and UV crosslink at 254 nm can stably enhance the detection level of PAR. However, the limitation of this assay is that the size of PAR detectable using the 10H antibody must be around 20 ADP-ribose residues, since the antibody cannot bind PAR of lower size.

Suggested Citation

  • Yuko Kudo & Yuka Sasaki & Takae Onodera & Jun Hashimoto & Tadashige Nozaki & Kenji Tamura & Masatoshi Watanabe & Mitsuko Masutani, 2018. "Measurement of Poly(ADP-ribose) Level with Enhanced Slot Blot Assay with Crosslinking," Challenges, MDPI, vol. 9(2), pages 1-10, July.
    • Handle: RePEc:gam:jchals:v:9:y:2018:i:2:p:27-:d:155780
    as

    Download full text from publisher

    File URL: https://www.mdpi.com/2078-1547/9/2/27/pdf
    Download Restriction: no
    File URL: https://www.mdpi.com/2078-1547/9/2/27/
    Download Restriction: no
    ---><---

    Corrections

    All material on this site has been provided by the respective publishers and authors. You can help correct errors and omissions. When requesting a correction, please mention this item's handle: RePEc:gam:jchals:v:9:y:2018:i:2:p:27-:d:155780. See general information about how to correct material in RePEc.

    If you have authored this item and are not yet registered with RePEc, we encourage you to do it here. This allows to link your profile to this item. It also allows you to accept potential citations to this item that we are uncertain about.

    We have no bibliographic references for this item. You can help adding them by using this form .

    If you know of missing items citing this one, you can help us creating those links by adding the relevant references in the same way as above, for each refering item. If you are a registered author of this item, you may also want to check the "citations" tab in your RePEc Author Service profile, as there may be some citations waiting for confirmation.

    For technical questions regarding this item, or to correct its authors, title, abstract, bibliographic or download information, contact: MDPI Indexing Manager (email available below). General contact details of provider: https://www.mdpi.com .

    Please note that corrections may take a couple of weeks to filter through the various RePEc services.

    IDEAS is a RePEc service. RePEc uses bibliographic data supplied by the respective publishers.