Development of high-resolution pulse-chase method by use of click chemical reaction

将带有烷基或叠氮基的前体分子培养的细胞快速冷冻，并将冷冻断裂复制品进行点击化学反应以标记膜脂质和蛋白质用于电子显微镜。前体处理后，磷脂酰胆碱的标记以时间依赖性的方式增加。在芽殖酵母中，内质网、核膜、胞囊和液泡中的标记密度在两个膜小叶中是相等的，而在质膜中的标记密度表现出明显的不对称性。

Cells cultured with precursor molecules bearing an alkyl or an azido group were quick-frozen and freeze-fracture replicas were subjected to click chemical reaction to label membrane lipids and proteins for electron microscopy. The labeling for phosphatidylcholine increased in a time-dependent manner after the precursor treatment. In budding yeast, the labeling density in the endoplasmic reticulum, nuclear membrane, mitochondrion, and vacuole was equivalent in the two membrane leaflets, whereas that in the plasma membrane showed clear asymmetry.