A fluorescence image study of cobra phospholipase A2 action on fluid-state lipid bilayers
碩士 === 國立中正大學 === 化學研究所 === 99 === The real cell membrane is fluid. We therefore would like to study the interactions of PLA2 with fluid-state lipid membrane. The challenge is that lipid molecules in the fluid-state membrane diffusion very fast, leading to blurred images. So we first need to prepare...
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| Format: | Others |
| Language: | zh-TW |
| Online Access: | http://ndltd.ncl.edu.tw/handle/00391465189781306494 |
| Summary: | 碩士 === 國立中正大學 === 化學研究所 === 99 === The real cell membrane is fluid. We therefore would like to study the interactions of PLA2 with fluid-state lipid membrane. The challenge is that lipid molecules in the fluid-state membrane diffusion very fast, leading to blurred images. So we first need to prepare fluid but isolated micro-sized membranes to observe the PLA2 actions on membranes.
According to literature the isolate membrane can be constructed using photolithography or scratch methods. But they are no use in our experiment. We choose air bubble strip to make isolated membrane. A 10 µL air bubble touches membrane and removes patches of membrane. Isolated and fluid membrane would be found in the defect area.
The isolated and fluid membranes were prepared by the bubble stripping method. We found that PLA2 actions on the fluid membrane exhibited similar behaviors as on the gel-state membrane. PLA2 preferred the adsorption on the edge of the membrane where the curvature is high. After a lag period, the PLA2 action bursts. PLA2 acts more effectively on the fluid membrane than on the gel-state membrane.
Using air bubble strip to form isolated membrane is difficult. Therefore we used membrane fluorescent probe FA to detect small unilamellar vesicles(SUV) and PLA2 interaction. We can see fluorescence change after the hydrolysis of SUV.
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