Mannik Jaana, Meyers Alex, Dalhaimer Paul
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee.
Department of Chemical and Biomolecular Engineering, University of Tennessee.
J Vis Exp. 2014 Apr 1(86):50981. doi: 10.3791/50981.
Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method-- density gradient centrifugation--is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques. In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins. In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions. The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.
脂滴是一种动态细胞器，存在于大多数真核细胞和某些原核细胞中。从结构上看，脂滴由中性脂质核心和磷脂单层膜组成。确定脂滴细胞功能最有用的技术之一是对结合蛋白进行蛋白质组学鉴定，这些蛋白可与脂滴一起分离出来。本文描述了两种从两种不同的真核生物（裂殖酵母和人胎盘绒毛细胞）中分离脂滴及其结合蛋白的方法。尽管这两种技术存在差异，但两种制备方法都采用了主要方法——密度梯度离心法。这表明所介绍的脂滴分离技术具有广泛的适用性。在第一个方案中，通过酶解细胞壁将酵母细胞转化为原生质球。然后将所得原生质球在松配合匀浆器中轻轻裂解。向裂解液中加入聚蔗糖以提供密度梯度，混合物离心三次。第一次离心后，脂滴与内质网（ER）、质膜和液泡一起位于离心管的白色漂浮层中。随后的两次离心用于去除其他三种细胞器。结果得到的一层仅含有脂滴和结合蛋白。在第二个方案中，通过用胰蛋白酶和DNA酶I进行酶解从足月人胎盘中分离出胎盘绒毛细胞。细胞在松配合匀浆器中匀浆。通过低速和中速离心步骤去除未破碎的细胞、细胞碎片、细胞核和线粒体。向匀浆中加入蔗糖以提供密度梯度，混合物离心以将脂滴与其他细胞组分分离。两种方案中脂滴的纯度均通过蛋白质免疫印迹分析得到证实。两种制备方法得到的脂滴组分均适用于后续的蛋白质组学和脂质组学分析。
