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Isolation of Sarcolemmal Plasma Membranes by Mechanically Skinning Rat Skeletal Muscle Fibers for Phospholipid Analysis
Author(s) -
Fajardo Val Andrew,
McMeekin Lauren,
Basic Admir,
Lamb Graham D.,
Murphy Robyn M.,
LeBlanc Paul J.
Publication year - 2013
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-013-3770-x
Subject(s) - endoplasmic reticulum , membrane , chemistry , skeletal muscle , sarcolemma , biochemistry , sphingomyelin , biophysics , anatomy , biology
Membrane phospholipid (PL) composition has been shown to affect cellular function by altering membrane physical structure. The sarcolemma plasma membrane (SL pm ) is integral to skeletal muscle function and health. Previous studies assessing SL pm PL composition have demonstrated contamination from transverse (t)‐tubule, sarcoplasmic reticulum, and nuclear membranes. This study assessed the possibility of isolating SL by mechanically skinning skeletal muscle fiber segments for the analysis of SL pm PL composition. Mechanically skinned SL pm from rat extensor digitorum longus (EDL) muscle fibers underwent Western blot analysis to assess contamination from t‐tubule, sarcoplasmic reticulum, nuclear and mitochondrial membranes. The results indicate that isolated SL pm had minimal nuclear and mitochondrial membrane contamination and was void of contamination from sarcoplasmic reticulum and t‐tubule membranes. After performing both high‐performance thin layer chromatography and gas chromatography, we found that the SL pm obtained by mechanical skinning had higher sphingomyelin and total fatty acid saturation and lower phosphatidylcholine when compared to previous literature. Thus, by avoiding the use of various chemical treatments and membrane fractionation, we present data that may truly represent the SL pm and future studies can use this technique to assess potential changes under various perturbations and disease conditions such as insulin resistance and muscular dystrophy.