I want to isolate crude mitochondria from primary myoblast cells for proteomics studies. The interesting thing is that I can enrich mitochondrial fraction without cytoplasmic contamination in C2C12 cells. However, in the primary myoblast case, although the mitochondrial fraction is enriched, I couldn't decrease cytosolic protein contamination. Different parameters that I've changed are as follows:
- cell lysis/homogenization method (Dounce homogenizer, syringe, sonication)
- centrifuge speed: [(first centrifuge: 800xg, 1200xg, 2000xg), (second centrifuge: 10000xg, 11000xg, 14000xg, 18000xg)]
- cell type: primary myoblast or differentiated primary myotubes
- cell number: two T75 flask, three T75 flask
- cell detachment method: trypsin or cell scraper
The isolation buffer that I used is as follows:
•50mM Tris-HCl pH 7.4
•0.075 M Sucrose
•0.225 M Mannitol
•0.1 M KCl
•0.2% Fatty acid-free BSA
•Protease inhibitor cocktail
•Phosphatase inhibitor (SO)
P.S. I've also tried Qproteome Mitochondria Isolation Kit and Mitochondria Isolation Kit for Cultured Cells (Thermo Scientific). However, I couldn't get rid of cytoplasmic proteins in the mitochondrial fraction.
The Qproteome mitochondria isolation kit offers the possibility to further purify the mitochondrial fraction by a percol/ficol gradient. This step should minimize cytosolic contaminations.
Gisela Beutner thank you for your answer. However, I need to mention that I got no mitochondrial pellet with the Qproteome mitochondria isolation kit. Even if I got a mitochondrial pellet with differential centrifugation (without using a kit), I cannot apply gradient centrifugation since I work with human primary myoblast cells and the ficoll/ percoll gradient method needs so many cells (like 100 million cells) as the starting material.
James B Stewart thank you for your answer. Actually, I used an anti-TOMM20 antibody as the mitochondrial marker and I got a strong signal. My problem is that I also got GAPDH signal in the mitochondrial fraction.
while in theory, it seems possible, due to the very high sensitivity of MS/MS some cytoplasmic protein will be always detected. You can try it yourself - make several repetitive centrifugation, resuspension, dilution. Then in the washed prep check the presence of cytoplasmic proteins.
also, try to omit protease inhibitor cocktail
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