Armstrong J M, Newman J D
Arch Biochem Biophys. 1985 May 1;238(2):619-28. doi: 10.1016/0003-9861(85)90207-3.
A method for the preparation of plasma membrane fractions from liver is described. A crude membrane fraction, substantially free of mitochondria and nuclei, is collected by centrifuging liver homogenate over a pad of 1.45 M sucrose. This interfacial fraction is further purified by isopycnic centrifugation in a self-generating density gradient of Percoll. All steps are carried out in buffered, isotonic solutions. Membrane prepared in this way is enriched in marker enzymes associated with plasma membrane, while marker enzymes for cellular components other than plasma membrane are diminished. Contamination by mitochondrial components is very much less than for membrane prepared from hypotonic liver homogenates. The membrane fraction also shows specific insulin binding, and negligible degradation of insulin. The procedure is rapid, uses simple equipment, and can be completed within 3 h.
本文描述了一种从肝脏制备质膜组分的方法。通过在1.45M蔗糖垫层上离心肝脏匀浆，收集基本不含线粒体和细胞核的粗膜组分。该界面组分通过在Percoll自生成密度梯度中进行等密度离心进一步纯化。所有步骤均在缓冲等渗溶液中进行。以这种方式制备的膜富含与质膜相关的标记酶，而质膜以外的细胞成分的标记酶则减少。线粒体成分的污染比从低渗肝脏匀浆制备的膜要少得多。该膜组分还显示出特异性胰岛素结合，且胰岛素降解可忽略不计。该方法快速，使用简单设备，可在3小时内完成。
