Measurement of Poly(ADP-ribose) Level with Enhanced Slot Blot Assay with Crosslinking
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 PA...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2018-07-01
|
| Series: | Challenges |
| Subjects: | |
| Online Access: | http://www.mdpi.com/2078-1547/9/2/27 |
| _version_ | 1819064148847230976 |
|---|---|
| author | Yuko Kudo Yuka Sasaki Takae Onodera Jun Hashimoto Tadashige Nozaki Kenji Tamura Masatoshi Watanabe Mitsuko Masutani |
| author_facet | Yuko Kudo Yuka Sasaki Takae Onodera Jun Hashimoto Tadashige Nozaki Kenji Tamura Masatoshi Watanabe Mitsuko Masutani |
| author_sort | Yuko Kudo |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T15:25:58Z |
| format | Article |
| id | doaj.art-a32d8b768bc2433681828d75a9946a91 |
| institution | Directory Open Access Journal |
| issn | 2078-1547 |
| language | English |
| last_indexed | 2024-12-21T15:25:58Z |
| publishDate | 2018-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Challenges |
| spelling | doaj.art-a32d8b768bc2433681828d75a9946a912022-12-21T18:58:54ZengMDPI AGChallenges2078-15472018-07-01922710.3390/challe9020027challe9020027Measurement of Poly(ADP-ribose) Level with Enhanced Slot Blot Assay with CrosslinkingYuko Kudo0Yuka Sasaki1Takae Onodera2Jun Hashimoto3Tadashige Nozaki4Kenji Tamura5Masatoshi Watanabe6Mitsuko Masutani7Genome Stability Research Division, Lab of Collaborative Research, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, JapanDivision of Cell Signaling, Lab of Collaborative Research, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, JapanDivision of Cell Signaling, Lab of Collaborative Research, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, JapanDepartment of Breast and Medical Oncology, National Cancer Center Hospital, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, JapanDepartment of Pharmacology, Faculty of Dentistry, Osaka Dental University, 8-1, Kuzuhahanazono-cho, Hirakata, Osaka 573-1121, JapanDepartment of Breast and Medical Oncology, National Cancer Center Hospital, Tsukiji 5-1-1, Chuo-ku, Tokyo 104-0045, JapanDivision of Materials Science & Chemical Engineering, Graduate School of Engineering, Yokohama National University, 79-5, Tokiwadai, Hodogaya-ku, Yokohama 240-8501, JapanGenome Stability Research Division, Lab of Collaborative Research, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, JapanPoly(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.http://www.mdpi.com/2078-1547/9/2/27poly(ADP-ribose)PARP inhibitorpharmacodynamic assayUV crosslink |
| spellingShingle | Yuko Kudo Yuka Sasaki Takae Onodera Jun Hashimoto Tadashige Nozaki Kenji Tamura Masatoshi Watanabe Mitsuko Masutani Measurement of Poly(ADP-ribose) Level with Enhanced Slot Blot Assay with Crosslinking Challenges poly(ADP-ribose) PARP inhibitor pharmacodynamic assay UV crosslink |
| title | Measurement of Poly(ADP-ribose) Level with Enhanced Slot Blot Assay with Crosslinking |
| title_full | Measurement of Poly(ADP-ribose) Level with Enhanced Slot Blot Assay with Crosslinking |
| title_fullStr | Measurement of Poly(ADP-ribose) Level with Enhanced Slot Blot Assay with Crosslinking |
| title_full_unstemmed | Measurement of Poly(ADP-ribose) Level with Enhanced Slot Blot Assay with Crosslinking |
| title_short | Measurement of Poly(ADP-ribose) Level with Enhanced Slot Blot Assay with Crosslinking |
| title_sort | measurement of poly adp ribose level with enhanced slot blot assay with crosslinking |
| topic | poly(ADP-ribose) PARP inhibitor pharmacodynamic assay UV crosslink |
| url | http://www.mdpi.com/2078-1547/9/2/27 |
| work_keys_str_mv | AT yukokudo measurementofpolyadpriboselevelwithenhancedslotblotassaywithcrosslinking AT yukasasaki measurementofpolyadpriboselevelwithenhancedslotblotassaywithcrosslinking AT takaeonodera measurementofpolyadpriboselevelwithenhancedslotblotassaywithcrosslinking AT junhashimoto measurementofpolyadpriboselevelwithenhancedslotblotassaywithcrosslinking AT tadashigenozaki measurementofpolyadpriboselevelwithenhancedslotblotassaywithcrosslinking AT kenjitamura measurementofpolyadpriboselevelwithenhancedslotblotassaywithcrosslinking AT masatoshiwatanabe measurementofpolyadpriboselevelwithenhancedslotblotassaywithcrosslinking AT mitsukomasutani measurementofpolyadpriboselevelwithenhancedslotblotassaywithcrosslinking |