What are the most popular methods used for large-scale mitochondrial isolation?

Differential centrifugation (DC) and density gradient centrifugation (DGC) are commonly techniques employed to separate organelles (such as mitochondria) based on their mass and sedimentation features. In DGC, the cell or tissue homogenate undergoes centrifugation through progressively denser media bands. While DGC yields a purer fraction, it results in a lower mitochondrial yield compared to DC. However, detergents and adjustments made to homogenization and centrifugation speed can increase yield.

Affinity purification (AP) involves capturing mitochondria by binding mitochondrial surface proteins antibodies conjugated to magnetic beads, which are held in place within a magnetic field. AP provides a high yield and purity and also enables the isolation of specific mitochondrial subspecies without the need for centrifugation. 

Additionally, it has the advantages of low sample requirement and is time-efficient. Another technique is fluorescence activated organelle sorting (FAOS). After detecting a fluorescent label, such as reporter genes or dyes, FAOS isolates mitochondria by assigning a specific charge to individual droplets containing mitochondria. This effectively separates them from other organelles or mitochondrial samples. FAOS allows the isolation of functional and purified mitochondrial samples, leading to a higher yield while reducing the need for the initial material. 

Capillary electrophoresis (CE), field-flow fractionation (FFF), and free-flow electrophoresis (FFE), techniques are all utilized to separate organelle subspecies based on their sedimentary features. In these methods, samples move along a channel through laminar flow. In CE and FFE, organelles are separated by their isoelectric point using an electric field, while FFF utilizes a perpendicular cross flow to separate molecules based on mass and size. FFE is more time-efficient and yields a purer product, although it forms a smaller yield. However, it comes with higher equipment costs in comparison to DGC.