For purification of Tom20 and Tom22, wild-type or indicated variants, cell debris was collected during mitochondria isolation (described above) and 1% digitonin was added to extract protein. The suspension was centrifuged at 150,000 × g for 30 min at 4 °C and then the supernatant was incubated with Flag-affinity beads (Sigma) for 1 h at 4 °C. The resin was washed in a gravity column by washing buffer (20 mM MOPS pH 7.4, 150 mM KCI, 10% [vol/vol] glycerol, 0.01% digitonin) with 10 column volumes. Proteins were eluted with Flag peptides (Genscript) and concentrated to 100 μL using a 20-kDa cutoff centrifugal filter (Millipore) and analyzed by SDS-PAGE. Truncated Tom22 proteins were obtained by treating with TEV protease to remove different parts. All proteins were further purified by gel filtration with a Superose 200 Increase 3.2/300 GL column (GE Healthcare) and the fractions were collected.
For Su9-TEV-DHFR-EGFP, the 293F cells were collected and extracted by 1% digitonin. After centrifugation, supernatant was incubated with Strep-affinity beads (Sigma). Su9-TEV-DHFR-EGFP was eluted with buffer (20 mM MOPS pH 7.4, 150 mM KCI) containing 5 mM desthiobiotin (Sigma). Part of the protein was treated with TEV protease to remove Su9. All proteins were further purified by gel filtration with a Superose 200 Increase 3.2/300 GL column (GE Healthcare) and the fractions were collected.
For Su9-TEV-DHFR-EGFP, the 293F cells were collected and extracted by 1% digitonin. After centrifugation, supernatant was incubated with Strep-affinity beads (Sigma). Su9-TEV-DHFR-EGFP was eluted with buffer (20 mM MOPS pH 7.4, 150 mM KCI) containing 5 mM desthiobiotin (Sigma). Part of the protein was treated with TEV protease to remove Su9. All proteins were further purified by gel filtration with a Superose 200 Increase 3.2/300 GL column (GE Healthcare) and the fractions were collected.