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

Danyi Zhou; Sheng Zhong; Xinyu Han; Dandan Liu; Hezhi Fang; Ya Wang

doi:10.1016/j.xpro.2023.102088

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

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

Authors

Danyi Zhou¹, Sheng Zhong¹, Xinyu Han¹, Dandan Liu¹, Hezhi Fang¹, Ya Wang²

Affiliations

¹ Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China.
² Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China. Electronic address: yawang@wmu.edu.cn.

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.

Keywords: Cell Membrane; Cell culture; Cell separation/fractionation; Protein Biochemistry.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Endopeptidase K
Membrane Proteins
Mitochondria*
Mitochondrial Proteins*
Octoxynol

Substances

Mitochondrial Proteins
Membrane Proteins
Octoxynol
Endopeptidase K