| Project/Area Number |
26840049
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Biophysics
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| Research Institution | Kyoto University |
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Principal Investigator |
PUJALS Silvia 京都大学, 物質-細胞統合システム拠点, 研究員 (10611866)
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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 |
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2015: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
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| Keywords | Bioimaging / Electron microscopy |
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| Outline of Final Research Achievements |
First, we established the conditions for an optimal electron microscopy (EM) cellular labeling by using a plasmid encoding for SNAP tag-β-2 adrenergic receptor protein. Then, we applied SNAP tag method to achieve the EM labeling for basal membrane sheets and intracellular proteins. We have used Caveolin-1 protein, as a model, as it has been widely studied in our lab. First, we successfully cloned Caveolin-1 gene into the SNAP-tag plasmid. Then we checked it by confocal microscopy, both with live and fixed cells and there was a nice punctuated labeling, characteristic of caveolae.
Thus, we assayed the SNAP tag system for the labeling of basal membrane sheets. After quick-freezing and freeze-drying the cells, we observed them by TEM to confirm the specific labeling of Caveolin-1 in caveolae. We had to find the optimal conditions for the labeling of unroofed cells. The most difficult part was to find a good nanoparticle-streptavidin conjugate with electron microscope quality.
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