Boone C W, Ford L E, Bond H E, Stuart D C, Lorenz D
J Cell Biol. 1969 May;41(2):378-92. doi: 10.1083/jcb.41.2.378.
A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20-50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30-33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 micromole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with (125)I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells.
本文描述了一种从HeLa细胞中分离质膜片段的方法。该过程始于制备细胞膜“空泡”，通过低渗肿胀细胞的温和破裂、反复洗涤去除大部分细胞内容物，并在不连续蔗糖密度梯度上分离空泡。然后将空泡在pH 8.6下进行最小限度的超声处理（5秒），这会使空泡膜收缩成小囊泡，但使任何剩余的较大细胞内颗粒保持完整，并可通过差速离心分离。然后将空泡膜囊泡在pH 8.6的三羟甲基氨基甲烷-镁缓冲液中的20-50% w/w连续蔗糖梯度上进行等密度离心。在30-33%（峰值密度 = 1.137）处回收了一条形态学上均匀的光滑囊泡带，主要来源于质膜。质膜部分含有每毫克1.5微摩尔无机磷/小时的钠钾激活ATP酶活性、3%的RNA，以及来自同一梯度中包含线粒体和粗面内质囊泡的较重部分的NADH-细胞色素c还原酶活性的13.8%。用（125）I标记的马抗体标记活HeLa细胞的质膜，并跟踪整个分离过程。质膜囊泡部分的特异性抗体结合比原始全细胞增加了49倍。
