CAUTION: MOPS, polyvinlypyrrolidone (see Appendix 3)
1. Follow Steps 1-7 in Chloroplast Preparation described above, but use Mito homogenization buffer in place of Xpl homogenization buffer.
2. Pellet chloroplasts by centrifuging in a Sorvall GSA rotor at 5500 rpm for 5 minutes.
3. Recover the supernatant.
4. Pellet mitochondria by centrifuging in a Sorvall SS-34 rotor at 13,000 rpm for 10 minutes.
5. Meanwhile, prepare a Percoll step gradient in an Oak Ridge tube by gently layering the following solutions, bottom to top, using a 10ml pipette: 6 ml of 60% (v/v) Percoll; 14 ml of 45% (v/v) Percoll; 8 ml of 27% (v/v) Percoll; 8 ml of 21% (v/v) Percoll. Keep on ice.
6. After centrifugation, carefully decant the supernatant. Use a paintbrush (wetted in Mito homogenization buffer) to gently resuspend the mitochondria in the residual buffer.
Optional: Wash pellet in Mito homogenization buffer.
7. Gently layer the resuspended mitochondrial pellet on top of the gradient and centrifuge in a Sorvall SS-34 rotor at 10,500 rpm for 30 minutes, with the brake off.
A faint brownish band may be visible in the lower third of the gradient; this is the mitochondrial band.
8. Use a PI000 automatic pipettor to remove 2-ml fractions from the top of the gradient.
The step gradient should smooth out during centrifugation (Schwitz-guebel and Siegenthaler 1984). In practice, the soluble proteins stay at the top, chloroplasts migrate to the upper portion of the gradient (fractions 2-6), and mitochondria end up in the lower third (fractions 12-15). There will be -17 2-ml fractions.
9. Pool the fractions containing mitochondria (usually fractions 11-15, with fraction 1 being the top of the gradient) in an Oak Ridge tube, and fill the tube with Mito homogenization buffer.
10. Pellet mitochondria by centrifuging in a Sorvall SS-34 rotor at 13,000 rpm for 10 minutes.
11. Determine enrichment of organelles as described above for chloro-plasts (Step 16, page 219).
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