Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting

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Konovalova , S 2019 , ' Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting ' , Journal of Visualized Experiments , no. 144 , 59269 . https://doi.org/10.3791/59269

Title: Analysis of Mitochondrial Respiratory Chain Complexes in Cultured Human Cells using Blue Native Polyacrylamide Gel Electrophoresis and Immunoblotting
Author: Konovalova, Svetlana
Contributor organization: Centre of Excellence in Stem Cell Metabolism
STEMM - Stem Cells and Metabolism Research Program
Henna Tyynismaa / Principal Investigator
Research Programme for Molecular Neurology
Research Programs Unit
University of Helsinki
Date: 2019-02
Language: eng
Number of pages: 7
Belongs to series: Journal of Visualized Experiments
ISSN: 1940-087X
DOI: https://doi.org/10.3791/59269
URI: http://hdl.handle.net/10138/301118
Abstract: Mitochondrial respiration is performed by oxidative phosphorylation (OXPHOS) complexes within mitochondria. Internal and environmental factors can perturb the assembly and stability of OXPHOS complexes. This protocol describes the analysis of mitochondrial respiratory chain complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) in application to cultured human cells. First, mitochondria are extracted from the cells using digitonin, then using lauryl maltoside, the intact OXPHOS complexes are isolated from the mitochondrial membranes. The OXPHOS complexes are then resolved by gradient gel electrophoresis in the presence of the negatively charged dye, Coomassie blue, which prevents protein aggregation and ensures electrophoretic mobility of protein complexes towards the cathode. Finally, the OXPHOS complexes are detected by standard immunoblotting. Thus, BN-PAGE is a convenient and inexpensive technique that can be used to evaluate the assembly of entire OXPHOS complexes, in contrast to the basic SDS-PAGE allowing the study of only individual OXPHOS complex subunits.
Subject: Biology
Issue 144
Mitochondria
OXPHOS complexes
blue native PAGE
electrophoresis
respiratory chain complexes
mitochondrial proteostasis
OXIDATIVE-PHOSPHORYLATION COMPLEXES
3111 Biomedicine
Peer reviewed: Yes
Usage restriction: openAccess
Self-archived version: publishedVersion


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