Mitochondrial Isolation: A Technique to Isolate Mitochondria from Human Ovarian Cancer Tissue Sample

Abstract

Source: Zhan, X. et al. Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis. J. Vis. Exp. (2019)

This video describes the procedure for isolating mitochondria from human ovarian cancer tissues using differential-speed centrifugation in combination with density gradient centrifugation. The isolated mitochondria can be used for proteomic analysis of human ovarian cancer mitochondrial proteome.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Preparation of mitochondria from human ovarian cancer tissues

Ovarian tissue samples including ovarian cancer tissues (n = 7) were used for this protocol.

1. Prepare 250 mL of the mitochondrial isolation buffer by mixing 210 mM mannitol, 70 mM sucrose, 100 mM potassium chloride (KCl), 50 mM Tris-HCl, 1 mM diamine tetraacetic acid (EDTA), 0.1 mM ethylene glycol bis(2....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Diamine tetraacetic acid (EDTA)	Sigma	798681-100G	Anhydrous, free-flowing, Redi-Dri, ≥98%
DTT	Sigma	10197777001	1,4-Dithiothreito
Ethylen glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA)	Sigma	E0396-10G	BioXtra, ≥97 .0%
Homogenizer	SilentShake	HYQ-3110
Low-temperature super-speed centrifuge	Eppendorf	5424R
Centrifuge	XiangYi	TDZ4--WS
Mannitol	Macklin	M813424-100G	Mannitol is a polyol (polyhydric alcohol) produced from hydrogenation from fructose that functions as a sweetener, humectant, and bulking agent. It has low hygroscopicity and poor oil solvency.
Phenylmethanesulfonyl fluoride (PMSF) protease inhibitor	Solarbio	P0100-1ML	PMSF is a protease inhibitor that reacts with serine residues to inhibit trypsin, chymotrypsin, thrombin, and papain.
Potassium chloride	Macklin	P816354-25G	Potassium chloride, KCI, also known as potassium muriate and sylvite, is a colorless crystalline solid with a salty taste that melts at 776°C (1420 OF). It is soluble in water, but insoluble in alcohol. Potassium chloride is used in fertilizers, pharmaceuticals, photography, and as a salt substitute