Author:
Anunciado-Koza Rea Victoria P.,Guntur Anyonya R.,Vary Calvin P.,Gartner Carlos A.,Nowak Madeleine,Koza Robert A.
Abstract
Abstract
Objective
Our goal was to isolate purified mitochondria from mouse skeletal muscle using a Percoll density gradient and to assess bioenergetic function and purity via Seahorse Extracellular Flux (XF) Analyses and mass spectrometry.
Results
Mitochondria isolated from murine quadriceps femoris skeletal muscle using a Percoll density gradient method allowed for minimally contaminated preparations with time from tissue harvest to mitochondrial isolation and quantification in about 3–4 h. Percoll purification from 100 to 200 mg fresh tissue yielded ~ 200–400 ug protein. Mitochondrial bioenergetics evaluated using the Seahorse XFe96 analyzer, a high-throughput respirometry platform, showed optimum mitochondrial input at 500 ng with respiratory control ratio ranging from 3.9 to 7.1 using various substrates demonstrating a high degree of functionality. Furthermore, proteomic analysis of Percoll-enriched mitochondria isolated from skeletal muscle using this method showed significant enrichment of mitochondrial proteins indicating high sample purity. This study established a methodology that ensures sufficient high quality mitochondria for downstream analyses such as mitochondrial bioenergetics and proteomics.
Publisher
Springer Science and Business Media LLC
Subject
General Biochemistry, Genetics and Molecular Biology,General Medicine