Identification of GPCR binding proteins by using a novel anti-FLAG antibody and magnetic nanobeads

• Project/Area Number: 23659157
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: General medical chemistry
• Research Institution: Juntendo University (2012); Kyushu University (2011)
• Principal Investigator: YOKOMIZO Takehiko 順天堂大学, 医学部, 教授 (60302840)
• Project Period (FY): 2010 – 2012
• Project Status: Completed (Fiscal Year 2012)
• Budget Amount: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000); Fiscal Year 2012: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2011: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: GPCR / 免疫沈降 / 結合タンパク質

Research Abstract

I established a novel anti-FLAG monoclonal antibody (2H8) with high-affinify and a low background. Combination of 2H8 antibody with a magnetic nanobeads enabled us to immunoprecipitate FLAG-tagged G-protein coupled receptors (GPCRs). Immunoprecipitated GPCRs were detectable by silver staining after separation by SDS-Page. I am analyzing some putative GPCR-binding proteins isolated by this novel immunoprecipitation techniques.

Research Products

• [Journal Article] A high-affinity monoclonal antibody against the FLAG tag useful for G-protein coupled receptor study (2012)
  • Author(s): Sasaki F, Okuno T, Saeki K, Liu Min, Onohara N, Kato H, Shimizu T, Yokomizo T
  • Journal Title: Anal. Biochem Volume: 425 Pages: 157-165
• [Journal Article] Helix 8 of leukotriene B4 receptor 1 inhibits ligand-induced internalization (2012)
  • Author(s): Aratake Y, Okuno T, Matsunobu T, Saeki K, Takayanagi R, Furuya S, Yokomizo T
  • Journal Title: FASEB J Volume: 26 Pages: 4068-4078
• [Journal Article] クローズアップ実験法:新規抗 FLAG 抗体 2H8 の活用法 (2012)
  • Author(s): 佐々木文之、横溝岳彦
  • Journal Title: 実験医学 Volume: 30 Pages: 2987-2992
• [Journal Article] A high-affinity monoclonal antibody against the FLAG tag useful for G-protein coupled receptor study (2012)
  • Author(s): F.Sasaki
  • Journal Title: Analytical Biochemistry Volume: (掲載決定) Issue: 2 Pages: 157-165
  • DOI: 10.1016/j.ab.2012.03.014
  • Peer Reviewed
• [Journal Article] クローズアップ実験法：新規抗FLAG抗体2H8の活用法 (2012)
  • Author(s): 佐々木文之、横溝岳彦
  • Journal Title: 実験医学 Volume: 30 Pages: 2987-2992
• [Journal Article] Leukotriene B_4 receptors : Novel roles in immunological regulations (2011)
  • Author(s): Yokomizo T
  • Journal Title: Adv.Enzyme Regul. Volume: 51 Issue: 1 Pages: 59-64
  • DOI: 10.1016/j.advenzreg.2010.08.002
  • Peer Reviewed
• [Journal Article] What is the natural ligand of GPR55? (2011)
  • Author(s): Okuno T, Yokomizo T
  • Journal Title: J.Biochem.(Tokyo) Volume: 149 Issue: 5 Pages: 495-497
  • DOI: 10.1093/jb/mvr022
  • NAID: 10030584968
  • Peer Reviewed
• [Presentation] A high-affinity monoclonal antibody against the FLAG tag useful for G-protein coupled receptor study (2013)
  • Author(s): F. Sasaki,, T. Yokomizo
  • Organizer: 5th Annual International Congress of Antibodies
  • Place of Presentation: 中国、杭州
• [Presentation] Generation of a high-affinity anti-FLAG monoclonal antibody 2H8 (2013)
  • Author(s): T. Yokomizo
  • Organizer: 5th Annual International Congress of Antibodies
  • Place of Presentation: 中国、杭州
  • Invited
• [Presentation] Expression and Function of a Leukotriene B4 receptor 1 in M2-type macrophage (2012)
  • Author(s): 佐々木文之, 古賀友紹, 佐伯和子, 奥野利明, 横溝岳彦
  • Organizer: 第41回日本免疫学会
  • Place of Presentation: 神戸
• [Presentation] Generation of a novel anti-FLAG monoclonal antibody. (2012)
  • Author(s): Yokomizo, T
  • Organizer: The 7th Korea-Japan Conference on Cellular Signaling for Young Scientists(招待講演)
  • Place of Presentation: Ulsan(韓国)
• [Presentation] Two distinct leukotriene B4 receptors: BLT1 and BLT2. (2011)
  • Author(s): Yokomizo, T, Liu, M, Saeki, K, Okuno, T
  • Organizer: The 10th JBS Biofrontier Symposium : New aspects of phospholipid biology and medicine(招待講演)
  • Place of Presentation: 福岡
• [Presentation] 生理活性脂質受容体の同定と生体内機能の解析・高親和性FLAG抗体の樹立と応用 (2011)
  • Author(s): 横溝岳彦
  • Organizer: 第2回九州エリア薬理・生理学若手研究会(招待講演)
  • Place of Presentation: 福岡
• [Presentation] Improved immunoprecipitation method for epitope-tagged GPCRs. (2011)
  • Author(s): Morisaki Y., Sasaki F., Aratake Y., Okuno T., Saeki K., Yokomizo T.
  • Organizer: The 10th JBS Biofrontier Symposium : New aspects of phospholipid biology and medicine
  • Place of Presentation: 福岡
• [Presentation] Improved immunoprecipitation method for epitope-tagged GPCRs. (2011)
  • Author(s): 森崎雄一, 佐々木文之, 荒武良総, 奥野利明, 佐伯和子, 横溝岳彦
  • Organizer: 第84回日本生化学会大会
  • Place of Presentation: 京都
• [Book] 「最新生理活性脂質研究-実験手法、基礎的知識とその応用-」遺伝子医学MOOK 24号 (2013)
  • Author(s): 横溝岳彦
  • Total Pages: 360
  • Publisher: メディカル ドゥ社
• [Book] 生化学実践問題 基礎と臨床をつなぐ420題 (2011)
  • Author(s): 横溝岳彦
  • Total Pages: 214
  • Publisher: 南江堂
• [Remarks] 新規 FLAG 抗体 2H8 の紹介
  • URL: http://plaza.umin.ac.jp/j_bio/Biochem1/2H8_Ab.html
• [Remarks] High affinity FLAG Ab 2H8-高親和性FLAG抗体2H8
  • URL: http://plaza.umin.ac.jp/j_bio/Biochem1/2H8_Ab.html
• [Remarks] 順天堂大学医学部第一生化学
  • URL: http://plaza.umin.ac.jp/j_bio/