Hope you've seen Araz Ostadi's answer in this forum:

https://www.researchgate.net/post/Can_anyone_give_me_the_protocol_or_video_about_how_to_separate_the_mitochondria_from_liver_tissue

To isolate good skeletal muscle mitochondria is not an easy task. First, you have to have some necessary equipment, which include Polytron tissue disintegrator, and ultracentrifuge for purification with Percoll gradient. The latter is important because without purification you will get a lot of damaged mitochondria. Polytron is necessary because muscle tissue is difficult to homogenize, and interfibrillar mitochondria are numerous but hidden deep between the muscle fibrils. The amount of subsarcolemmal mitochondria is small, and thus the yields will be very low. To receive more, much more numerous interfibrillar mitochondria, you have to use Nagarse or similar protease available at Sigma. And you have to be careful not to overdigest the tissue. There are numerous papers on the Nagarse method, but many important tricks are not discussed. I have published this year a book, “Practical Mitochondriology”, which you can find on Amazon.com (about or less than $25) where the method is described together with many tricks. Not that I advertise my book, but in the first hand, I wrote it to share my experience of 45 years practical work with mitochondria with young people like you. There are many things that affect the methods of mitochondria isolation, which are impossible to describe in a short post.

Hello, we are using (sligtly adapted) protocol developed by Rasmussen et al. - http://www.ncbi.nlm.nih.gov/pubmed/9324953. Instead of bacterial proteases we are working with trypsin (as trypsin together with collagenase is used for primary muscle cells isolation) - 2,5 % trypsin 3x diluted in ATP medium.