Enzyme Explorer


Mitochondria isolation is commonly utilized for apoptosis studies.1 Such studies are of central importance for the investigation of a number of major debilitating diseases including Parkinson’s disease and cancer.2,3 In addition, mitochondrial protein isolation is of importance in proteome studies.4,5

Different isolation procedures are required for mitochondria from "soft" tissues such as liver or brain, and from "hard" tissues such as skeletal muscle or heart muscle. The "soft" tissues are extracted in the presence of delipidated BSA that removes free fatty acids present in the tissue that cause uncoupling of respiration in the mitochondria.6 EGTA is also present in the buffer to chelate Ca2+ ions that cause mitochondrial swelling.

"Hard" tissues cannot be homogenized easily without pretreatment with a protease to promote breakdown of the cellular structure. The myofibrils in skeletal muscle tend to give a gelatinous consistency to the homogenate in non-ionic media (isotonic sucrose) and thus must be isolated in an ionic medium such as 100 mM MOPS, pH 7.5, containing 550 mM KCl and 5 mM EGTA.7

Mitochondria can be prepared easily from animal tissues by a simple method of homogenization followed by low (600 x g) and high speed (11,000 x g) centrifugation.8 The final pellet represents a crude mitochondrial fraction that may be used as the basis for further experiments. For a more purified "heavy" mitochondrial fraction that will be enriched with mitochondria as opposed to lysosomes and peroxisomes that normally contaminate this fraction, the low and high speed centrifugation steps can be changed to 1,000 x g and 3,500 x g, respectively.6

Assessment of the mitochondrial inner membrane integrity can be accomplished by testing of the electrochemical proton gradient (Dy) of the inner mitochondrial membrane.9 This may be achieved by measuring the uptake of the fluorescent carbocyanine dye JC-1 (Prod No. T4069) into the mitochondria.10,11 The outer membrane integrity may be measured by observing cytochrome c oxidase activity (using the Cytochrome c Oxidase Assay Kit, (Product No. CYTOC-OX1). This kit measures the activity in the presence and absence of the detergent n-dodecyl β-D-maltoside, and the ratio of the two activities provides a measure of the integrity of the outer membrane.

We offer a mitochondria isolation kit (Product No. MITO-ISO1) that includes trypsin and is sufficient for extraction of up to 10-20 g of animal tissue and 50 JC-1 assays of 2 ml. This kit enables the fast and easy isolation of an enriched mitochondrial fraction from animal tissues. Most of the isolated mitochondria will contain intact inner and outer membranes.

Proteases for Mitochondria Isolation

Nagarse (Protease, Bacterial)
Amino acid analysis and isoelectric focusing electrophoresis consistent with subtilisin Carlsberg.
Essentially free of DNase and Rnase
Reported useful for the isolation of hepatic,12 skeletal muscle13 and thyroid14 mitochondria
Prod No. P8038
Sublilisin A
Essentially free of DNase and Rnase
Reported useful for the isolation of hepatic15 and cardiac16 mitochondria
Prod No. P5380
Papain from papaya latex
Reported useful for the isolation of hepatic mitochondria.12
Prod No. P4762
Trypsin from porcine pancreas
Reported useful for the isolation of hepatic17 and mung bean18,19 mitochondria.
Prod No. T0303
Proteinase K
Reported useful for the isolation of hepatic,20 yeast,21 and mung bean18 mitochondria
Prod No. P6556


  1. Rampino, N., et al., Science, 275, 9679 (1997).
  2. Wallace, D.C., Novartis Foundation Symposium, 235, 247 (2001).
  3. Colin A. and Seamus M.J., Trends in Biochem. Sci., 26, 390 (2001).
  4. Lopez M.F., et al, Electrophoresis, 21, 3427 (2000).
  5. Rabilloud, T., et al, Electrophoresis, 19, 1006 (1998).
  6. Graham, J.M., Methods in Molecular Biology, 19: Biomembrane Protocols, Graham, J.M. and Higgins, J.A. (Eds.), pp 29-57(Humana Press, 1993)
  7. Lee, C.P., Biochem. Biophys. Acta, 1271, 21 (1995).
  8. Storrie, B. and Madden, E.A., Methods Enzymol., 182, 203 (1990).
  9. Gross, A., et al., J. Biol. Chem., 274, 1156 (1999).
  10. Reers, M., et al., Biochem., 30, 4480 (1991).
  11. Salvioli, S., et al., FEBS Letts., 411, 77 (1997).