What are the limitations of centrifugation-based methods of mitochondrial isolation?

There are various limitations of centrifugation-based methods of mitochondrial isolation. 

• One limitation is the potential risk of damaging mitochondria during the homogenization and isolation process. 
• Another drawback is the potential loss or modifications in ETC complexes, as certain subunits of mitochondrial electron transport chain complexes may be lost or altered during the centrifugation and homogenization steps. 
• A third limitation is that this procedure requires large quantities of tissue specimens for effective mitochondrial isolation. 
• Additionally, when using capillary electrophoresis-laser-induced fluorescence (CE-LIF), it has the limitation of mitochondria retention within the fine capillary tube, impacting yield. 
• Lastly, the homogenization and centrifugation steps may lead to an increased production of reactive oxygen species, which could hinder mitochondrial function.