Measurement of Poly(ADP-ribose) Level with Enhanced Slot Blot Assay with Crosslinking
Yuko Kudo,
Yuka Sasaki,
Takae Onodera,
Jun Hashimoto,
Tadashige Nozaki,
Kenji Tamura,
Masatoshi Watanabe,
Mitsuko Masutani
Affiliations
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
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
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
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
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
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.
