Steck T L, Lavasa M
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Anal Biochem. 1994 Nov 15;223(1):47-50. doi: 10.1006/abio.1994.1544.
A general method for isolating plasma membranes is described. Vegetative amoebae of Dictyostelium discoideum were allowed to directly adsorb raw colloidal gold of particle diameter 10-20 nm. After quenching the gold surface, the cells were lysed and the lysates were diluted in 60% sucrose and centrifuged through a 65% sucrose cushion to selectively pellet the gold-laden membranes. Three generally applicable exogenous cell surface markers were used to follow the plasma membranes: intercalated [3H]cholesterol, octadecylrhodamine, and the adsorbed gold colloid itself. The isolates routinely contained approximately 60% of these tags, enriched approximately 15-fold with respect to protein. The recovery and degree of enrichment of contaminating markers in the plasma membrane fraction were lysosomes (3% and 1-fold); mitochondria (11% and 3-fold); rough endoplasmic reticulum, as reflected by RNA (3% and 0.7-fold); and DNA (9% and 4-fold). Membrane proteins and lipids were quantitatively solubilized from the gold by detergents. We conclude that this methodology provides an approach to the isolation of plasma membranes which compares favorably to existing techniques with respect to yield, purity, and ease.
本文描述了一种分离质膜的通用方法。将盘基网柄菌的营养性变形虫直接吸附直径为10 - 20 nm的粗胶体金。在淬灭金表面后，裂解细胞，将裂解物在60%蔗糖中稀释，并通过65%蔗糖垫层离心，以选择性地沉淀负载金的膜。使用三种普遍适用的外源性细胞表面标记物来追踪质膜：插入的[3H]胆固醇、十八烷基罗丹明和吸附的金胶体本身。分离物通常含有约60%的这些标记物，相对于蛋白质富集约15倍。质膜部分中污染标记物的回收率和富集程度分别为：溶酶体（3%和1倍）；线粒体（11%和3倍）；由RNA反映的糙面内质网（3%和0.7倍）；以及DNA（9%和4倍）。膜蛋白和脂质可通过去污剂从金中定量溶解。我们得出结论，该方法在产量、纯度和简便性方面与现有技术相比，为质膜的分离提供了一种更好的方法。
