Quantitative retention of membrane lipids in the freeze-fracture replica
Quantitative retention of membrane lipids in the freeze-fracture replica
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DOI：
10.1007/s00418-007-0341-3
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发表时间：
2007-11-01
影响因子：
2.3
通讯作者：
Fujimoto, Toyoshi
中科院分区：
文献类型：
--
作者：
Fujita, Akikazu;Fujimoto, Toyoshi
SDS 消化的冷冻断裂复制品已被用作免疫电子显微镜的底物，以确定膜蛋白和脂质的定位。我们以及其他人都注意到，通过首先蒸发碳制备的复制品比先蒸发铂/碳然后再蒸发碳的传统制备方法更有效地进行标记。在本研究中，我们检查了碳优先复制品中的优异标记是否是由于 SDS 消化过程中膜分子的更好保留造成的。我们使用磷脂酰胆碱脂质体作为模型样品，并测量了 SDS 消化的复制品中保留的无机磷的量。结果表明，不同方法制备的复制品对无机磷酸盐的保留率相当，表明复制品上的标记强度与保留率不相关。有趣的是，尽管保留率相似，但单独由碳制成的复制品对神经节苷脂 GM1 和磷脂酰胆碱的标记远少于由碳和铂/碳制备的复制品。这些结果表明，与铂捕获的探针相比，探针可以更有效地与碳捕获的脂质结合。尽管如此，碳之后铂的蒸发对于正确的标记是必不可少的。
SDS-digested freeze-fracture replicas have been used as a substrate for immunoelectron microscopy to determine localization of membrane proteins and lipids. We, as well as others, have noticed that replicas prepared by first evaporating carbon are labeled more efficiently than conventional preparations in which platinum/carbon is evaporated first followed by carbon. In the present study, we examined whether the superior labeling in the carbon-first replica is caused by better retention of membrane molecules during SDS digestion. We used phosphatidylcholine liposomes as a model sample and measured the amount of inorganic phosphorus retained in the SDS-digested replica. The result showed that there was equivalent retention of inorganic phosphate among replicas prepared in different ways, indicating that labeling intensity on the replica did not correlate with the retention ratio. Interestingly, despite a similar retention ratio, replicas made of carbon alone gave far less labeling for ganglioside GM1 and phosphatidylcholine than replicas prepared by carbon followed by platinum/carbon. These results suggest that probes can bind to lipids captured by carbon more efficiently than those captured by platinum. Nonetheless, evaporation of platinum after carbon is indispensable for proper labeling.