| Project/Area Number |
26670812
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
Functional basic dentistry
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| Research Institution | Health Sciences University of Hokkaido |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
Morita Takao 北海道医療大学, 歯学部薬理学分野, 講師 (20326549)
Nezu Akihiro 北海道医療大学, 歯学部薬理学分野, 講師 (00305913)
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| Co-Investigator(Renkei-kenkyūsha) |
Shuto Satosh 北海道大学, 大学院薬学研究院・創薬有機化学研究室, 教授 (70241346)
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| Project Period (FY) |
2014-04-01 – 2016-03-31
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| Project Status |
Completed (Fiscal Year 2015)
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| Budget Amount *help |
¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2015: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Keywords | イメージング / カルシウム / イノシトール三リン酸 / アセチルコリン / 蛍光センサー / FRET / 細胞内シグナル |
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| Outline of Final Research Achievements |
We developed the CFLA-IP3 (Competitive Fluorescent Ligand Assay for IP3) as a conceptually new method to measure IP3 concentrations. The method is based on the combination of two rationally designed fluorescent molecules, low-affinity fluorescent Adenophostin A analogue (FLL) and a fluorescent IP3-binding protein (CFP-coupled LBD). Binding of the fluorescent ligands to the fluorescent IP3 sensor protein-expressing cells caused fluorescence resonance energy transfer (FRET). This principle was extended to a cell-free assay system using fluorescent IP3 sensor protein-bound agarose beads. The effect of FLL on the FRET signal of fluorescent IP3 sensor protein-bound beads was reduced by the subsequent addition of IP3. This method allowed quantitative measurement of IP3 concentrations as low as 10 nM and was applied to measure cytosolic IP3 concentrations in COS-7 cells.
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