线粒体分离及亚细胞定位检测法用于线粒体蛋白分析的实验方案

Protocol for mitochondrial isolation and sub-cellular localization assay for mitochondrial proteins.

机构信息

Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China.

出版信息

STAR Protoc. 2023 Mar 17;4(1):102088. doi: 10.1016/j.xpro.2023.102088. Epub 2023 Feb 3.

DOI:10.1016/j.xpro.2023.102088

PMID:36853693

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9932554/

Abstract

Here, we provide a protocol to isolate mitochondria from cultured cells and extract differently located mitochondrial proteins. We detail steps to separate both integral and peripheral membrane proteins from soluble proteins using sonication. We describe the separation of integral membrane proteins from the peripheral membrane and soluble proteins using sodium carbonate extraction. Furthermore, we detail the use of proteinase K and Triton X-100 to distinguish outer membrane proteins from mitochondrial proteins.

摘要

在这里，我们提供了一种从培养细胞中分离线粒体并提取不同位置线粒体蛋白的方案。我们详细介绍了使用超声处理分离整合膜蛋白和可溶性蛋白的步骤。我们还描述了使用碳酸钠提取法分离整合膜蛋白与外周膜和可溶性蛋白。此外，我们详细介绍了使用蛋白酶 K 和 Triton X-100 区分外膜蛋白和线粒体蛋白的方法。