Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation.
Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation.
复制标题
DOI：
10.1016/j.ab.2010.02.030
复制
发表时间：
2010-06-15
影响因子：
2.9
通讯作者：
Luschnig C
中科院分区：
文献类型：
--
作者：
Abas L;Luschnig C
微粒体膜组分的分离是涉及膜蛋白的研究中的常用程序。根据传统定义，微粒体膜是通过在超离心器中以100，000g离心线粒体后部分来收集的，这是最初为大量哺乳动物组织开发的方法。我们提出了一种从少量拟南芥植物材料中分离微粒体型膜的方法，该方法不依赖于超离心，而是使用微量离心机的较低相对离心力（21，000g）。我们表明，21，000 g沉淀物与100，000 g沉淀物相当，并且它含有常规微粒体组分中预期的所有膜组分。我们的方法结合了整个过程中的样品密度的特定操作，最小的预清除，最小体积的提取缓冲液，和最小的沉降路径长度。这些特征允许最大的膜产率，使得能够从有限量的材料中分离膜。我们进一步证明，传统的基于超离心的协议给次最大的产量由于损失在早期阶段的程序，也就是说，大量的微粒体类型的膜可以沉淀在典型的preclearance步骤过早。我们的协议避免了这种损失，从而确保最大的产量和代表性的总膜分数。我们的方法的原则可以适用于非植物材料。
Isolation of a microsomal membrane fraction is a common procedure in studies involving membrane proteins. By conventional definition, microsomal membranes are collected by centrifugation of a postmitochondrial fraction at 100,000g in an ultracentrifuge, a method originally developed for large amounts of mammalian tissue. We present a method for isolating microsomal-type membranes from small amounts of Arabidopsis thaliana plant material that does not rely on ultracentrifugation but instead uses the lower relative centrifugal force (21,000g) of a microcentrifuge. We show that the 21,000g pellet is equivalent to that obtained at 100,000g and that it contains all of the membrane fractions expected in a conventional microsomal fraction. Our method incorporates specific manipulation of sample density throughout the procedure, with minimal preclearance, minimal volumes of extraction buffer, and minimal sedimentation pathlength. These features allow maximal membrane yields, enabling membrane isolation from limited amounts of material. We further demonstrate that conventional ultracentrifuge-based protocols give submaximal yields due to losses during early stages of the procedure; that is, extensive amounts of microsomal-type membranes can sediment prematurely during the typical preclearance steps. Our protocol avoids such losses, thereby ensuring maximal yield and a representative total membrane fraction. The principles of our method can be adapted for nonplant material.
登录
查看更多内容
影响因子：
64.8
作者：
Jaillais, Yvon;Fobis-Loisy, Isabelle;Gaude, Thierry
通讯作者：
Gaude, Thierry
影响因子：
1.6
作者：
Kobae, Y;Mizutani, M;Maeshima, M
通讯作者：
Maeshima, M
影响因子：
6.9
作者：
Hanson, MR;Köhler, RH
通讯作者：
Köhler, RH
影响因子：
56.9
作者：
Claude, A.
通讯作者：
Claude, A.
影响因子：
7.4
作者：
BarPeled, M;Raikhel, NV
通讯作者：
Raikhel, NV