Isolation of Pure Mitochondria from Rat Kidneys and Western Blot of Mitochondrial Respiratory Chain Complexes

Cardiac, neuronal and renal tubular epithelial cells are the most metabolically active cells in the body. Their fate depends largely on their mitochondria as the primary energy generating system which participates in the control of apoptosis, cell cycle and metabolism. Thus, mitochondrial dysfunctio...

Full description

Saved in:

Bibliographic Details
Published in:	Bio-protocol Vol. 9; no. 19; p. e3379
Main Authors:	Micakovic, Tamara, Banczyk, Wiktoria Z, Clark, Euan, Kränzlin, Bettina, Peters, Jörg, Hoffmann, Sigrid Christa
Format:	Journal Article
Language:	English
Published:	United States Bio-Protocol 05-10-2019 Bio-protocol LLC
Subjects:	Biology Methods Diabetic nephropathy Mitochondria Renin angiotensin system Angiotensin II type 2 receptor Mitochondria isolation Western blotting
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	Cardiac, neuronal and renal tubular epithelial cells are the most metabolically active cells in the body. Their fate depends largely on their mitochondria as the primary energy generating system which participates in the control of apoptosis, cell cycle and metabolism. Thus, mitochondrial dysfunction is a hallmark of many chronic diseases including diabetic nephropathy. A drop in mitochondrial bioenergetics efficiency is often associated with altered expression of respiratory chain complexes. Moreover, recent studies demonstrate that cellular proteins can shuttle to mitochondria and modify their function directly. Here we illustrate two mitochondria isolation protocols; one is recommended if the purity of the mitochondrial fraction is a priority such as if the mitochondrial localization of a protein has to be validated, the other if a high yield of intact functional mitochondria is required for functional studies and quantitative Western blotting. Next, we provide a detailed protocol for Western blotting of isolated mitochondria and renal cortex either to prove the purity of isolated fractions or to quantify complexes of the mitochondrial respiratory chain. We used this approach to identify classically cell membrane bound angiotensin II receptors in mitochondria and to study the effect of these receptors on mitochondrial function in early stages of diabetic nephropathy.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2331-8325 2331-8325
DOI:	10.21769/BioProtoc.3379