In Vitro Reconstitution of the Endoplasmic Reticulum
In Vitro Reconstitution of the Endoplasmic Reticulum
复制标题
内质网的体外重建
DOI：
--
复制
发表时间：
2018
影响因子：
--
通讯作者：
M. Weiss
中科院分区：
文献类型：
--
作者：
Csilla Ferencz;Gernot Guigas;Andreas Veres;Brigitte Neumann;O. Stemmann;M. Weiss
体外细胞器的重建提供了在受控环境中进行定量和定性实验的可能性，而在活细胞中无法以相同的精度进行。继以前的报告，随后的协议列表描述了如何重建和量化的管状ER网络在体外的基础上纯化的微粒体从培养细胞和胞质溶胶从非洲爪蟾卵提取物。生物材料制备和重构分析主要需要基本的实验室仪器和化学品，并且可以在没有任何特定培训的情况下执行，这使得它们吸引了广泛的实验室。此外，为了促进明显更能反映体内环境的条件，该方法在文献中首次详细描述了从HeLa细胞中纯化微粒体。本文中的基本方案1描述了在不同基质上的重构过程，包括相关的荧光成像过程。ER微粒体和胞质溶胶的纯化（这两者都是该方法所需的）分别在支持方案1和2中详细描述。在支持方案3中描述了用聚丙烯酰胺凝胶涂覆表面。基本协议2概述了如何分割和分解ER网络的荧光图像，以及如何量化网络分支点之间的片段长度。所描述的定量评估提供了一个有意义的方法来分析细胞器结构的拓扑结构和几何形状。John Wiley & Sons，Inc.
Reconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to reconstitute and quantify a tubular ER network in vitro based on purified microsomes from culture cells and cytosol from Xenopus laevis egg extracts. Biological material preparation and reconstitution assays require mostly basic laboratory instrumentation and chemicals, and can be executed without any specific training, making them appealing to a wide range of laboratories. Moreover, to promote conditions that are markedly more reflective of in vivo environments, this method describes for the first time in the literature, the purification of microsomes from HeLa cells in some detail. Basic Protocol 1 in this article describes the reconstitution process on different substrates including the associated fluorescence imaging process. Purification of ER microsomes and cytosol, both of which are needed for this approach, are described in detail in Support Protocols 1 and 2, respectively. Coating of surfaces with polyacrylamide gels is described in Support Protocol 3. Basic Protocol 2 outlines how to segment and skeletonize fluorescence images of ER networks, and how to quantify segment lengths between the network's branching points. The described quantitative evaluation provides a meaningful approach to analyze the topology and geometry of organelle structures. © 2017 by John Wiley & Sons, Inc.
DOI：
10.1073/pnas.96.3.921
发表时间：
1999-02-02
影响因子：
11.1
作者：
Rotsch, C;Jacobson, K;Radmacher, M
通讯作者：
Radmacher, M
影响因子：
29
作者：
Radhakrishnan A;Goldstein JL;McDonald JG;Brown MS
通讯作者：
Brown MS
免疫球蛋白 G 重链和轻链的体外寡聚化。
DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Watkins,JD;Hermanowski,AL;Balch,WE
通讯作者：
Balch,WE