Superose 6 increase 3.2 300 gl column

Mitochondria were suspended with buffer B (20 mM MOPS pH 7.4, 150 mM KCI, 10% [vol/vol] glycerol, 1 mM PMSF) and mixed with 1% (vol/vol) digitonin (BIOSYNTH, No. D3200). After incubation for 3 h, the mixture was centrifuged at 150,000 × g for 30 min at 4 °C. Flag beads (Sigma) were added into the supernatant and incubated for 1 h at 4 °C. The beads were washed in a gravity column with 10 column volumes of washing buffer (20 mM MOPS pH 7.4, 150 mM KCI, 10% [vol/vol] glycerol, 0.01% digitonin). The TOM complex was eluted with washing buffer containing Flag peptides (Genscript) and concentrated to 100 μL using a 50-kDa cutoff centrifugal filter (Millipore). The complex was further purified by gel filtration with a Superose 6 Increase 3.2/300 GL column (GE Healthcare). Fractions were collected for sodium dodecyl sulfate polyAcrylamide gel electrophoresis (SDS-PAGE) analysis and cryosample preparation.

After purification with Flag beads (Sigma), the TOM complex was cross-linked by 0.1% glutaraldehyde (Sigma) for 1 h at 0 °C and the cross-linking reaction was quenched by 50 mM Tris⋅HCl (pH 8.0). The cross-linked complex was further purified by gel filtration with a Superose 6 Increase 3.2/300 GL column (GE Healthcare). Fractions were collected for SDS-PAGE analysis and concentrated to 20 μL using a 50-kDa cutoff centrifugal filter (Millipore) for the cryosample preparation.