I am isolating mitochondria from rat quadriceps, using the following procedure:

First I cut ~200 mg quadricep placed in 1 mL of ice-cold isolation buffer (Sucrose 100 mM, KCl 100 mM, Tris–HCl 50 mM, KH2PO4 1 mM, EGTA 0.1 mM, BSA 0.2%, at pH 7.4) for 4 min with a sharp scissor. Then I transfer the muscle pieces to 1 mL of the above-described isolation buffer containing 0.2 mg/mL bacterialproteinase (Type XXVII). After 2 min of incubation, I transfer the sample to a loosely fitting glass-Teflon homogenizer and carefully homogenize it for 2 min. After homogenization, I add 3 mL of protease free isolation buffer to the homogenate, which is then fractionated by centrifugation at 750g for 10 min at 4C. The samples is kept on ice during the whole procedure.

After the centrifugation (and sometimes even before) the homogenate often forms a gel-like structure. This results in a large viscous pellet that seems to bind the mitochondria (the mitochondria should be in the supernatant) resulting in much lower mitochondrial yield.

I have never had this problem when isolating mitochondria from liver, brain or heart and it also happens very rarely in skeletal muscle from human biopsies.

I would really appreciate hearing your suggestions and ideas of what I could do to avoid this gelation?