Begin by transferring one milliliter crude fraction of RAW 264.7 mitochondria into a 15-milliliter conical tube. Add 7 milliliters of salted mitochondrial buffer supplemented with 150 millimolar sodium chloride. To the suspension, add 50 microliters of TOM22 beads and then incubate the tube on a rotating wheel at low speed.
Place a column on the magnet and equilibrate the column with eight milliliters of salted mitochondrial buffer. Discard the flow-through. After sample incubation, transfer it to the column and discard the flow-through.
Next, wash the column three times with eight milliliters of salted mitochondrial buffer. Remove the column from the magnet and place it in a 15-milliliter conical tube. To elute the mitochondria, add 1.5 milliliters of salted mitochondrial buffer and immediately apply a plunger to elute the purified fraction into the tube.
Transfer the eluted fraction to a new 1.5-milliliter tube and perform centrifugation at 21, 000G for 10 minutes at 4 degrees Celsius. Discard the supernatant formed and store the brown pure mitochondrial pellet at 20 degrees Celsius for future studies. Immunoblot analysis of the purified mitochondrial extracts showed enrichment of mitochondrial citrate synthase and depletion of proteins from other cell organelles.
Mitochondrial integrity studies using electron microscopy revealed mitochondria with a classic oval shape and intact cristae surrounded by electron-dense antibody-coated beads. Further, proteome analysis showed enrichment of mitochondrial proteins and two distinct populations corresponding to mitochondrial and non-mitochondrial proteins.
