Isolation of mitochondria from Arabidopsis leaves (Keech et al., 2005)
Plant materials
Arabidopsis plants must be grown under short-day conditions as described in theliterature and harvested before the completion of the light cycle.
Preparation of Percoll gradient
1. Centrifuge a 50% Percoll solution (6 ml Percoll solution/13 ml PC tube x 6) at
39 000 g for 40 min in a Hitachi RPS40T-410 rotor (18,000rpm) and keep thedensity gradient thus produced at 4˚C before use.
After this centrifugation, the Percoll solution will form as a pellet at the bottomof the tube. Special care should be taken, at later steps, not to recover thepellet during Mit recovery.

Method B: to obtain purified mitochondria (Yield ~100-200 mg of totalmitochondrial proteins)
Grind approximately 50 g of leaves in 4 portions. All equipment should be chilled
at 4˚C and centrifuged at 4˚C.
1. Grind 12.5 g leaf tissue, using a mortar and pestle, in 15 ml of grinding buffer B
with a small amount (0.5 g) of quartz (fine granular, Merck).
2. Add 4 x ~20 ml of grinding buffer B. Combine all portions of homogenates andfilter through a nylon mesh (20 µm).
3. Centrifuge the extract (total ~200 ml) at 2,500 g for 5 min in a Hitachi RPR20-2
rotor (4,800 rpm, 4 ˚C) and centrifuge the resultant supernatant at 15,000 g for
15 min in an RPR20-2 rotor (11,500rpm, 4˚C).
4. Resuspend the pellet in ~750 µl of wash buffer B, using a small paintbrush, andvery gently homogenize twice, using a 5 ml glass homogenizer.
5. Carefully layer the resuspended mitochondrial preparation on top of the percoll
gradient (6 x 13PC tubes). The maximum load per gradient is the crude
mitochondria from g fresh leaves per ml gradient. Centrifuge for 15 min at
15,000 g in a Hitachi RPS40T-410 rotor (11,000rpm).
6. The mitochondria will form a whitish band close to the bottom of the tube. Thisband is aspirated and resuspended in wash buffer B to obtain (at least) a20-fold dilution. (Remove the major green band (broken thylakoids) byaspiration, and recover the mitochondria by pipetting).

7. Centrifuge at 15,000 g (4˚C) for 20 min in RPR20-2 rotor (11,500rpm).
Mitochondria should form a soft pellet. Repeat this last centrifugation step if thepellet is not sufficiently firm.
8. Resuspend the pellet in 200–300 µl of wash buffer B. [Option for respiratory
activity measurements: add four percent of the final volume of a stock solutionof protease inhibitor cocktail (Roche Applied Science)].
Tips:
・Do not use a washed Miracloth.
・Grind plant material thoroughly.
・Do not use too large a volume of percoll gradient (6 ml/12 g initial leaf tissue isenough). Also, be sure to use swing bucket rotors for the Percoll steps.
Grinding buffer B (100 ml):
0.3 M sucrose (mw 342.30)
60 mM TES-KOH, pH 8.0
1.0 M stock
2 mM EDTA-2Na·2H2O (mw. 372.24)
10 mM KH2PO4 (mw 136.09)
25mM tetrasodium pyrophosphate (mw 446.06)
1 mM glycine (mw 75.07)
1% (w/v) polyvinylpyrrolidone-40
1% (w/v) bovine serum albumin (BSA)
50 mM sodium ascorbate (mw 198.11)

10.269 g
6 ml(1.376g)
74.45 mg(0.4ml)
136.09 mg
1.115 g7.51 mg
1.0 g
1.0 g
990.55 mg

Plus 20 mM cysteine (mw 121.16, 242.32 mg) added just prior to grinding, andreadjustment of the pH to 8.0 with 1 M KOH.
Wash buffer B (100 ml):
0.3 M sucrose (mw 342.30)
10 mM TES (mw 229.25)
10 mM KH2PO4 (mw 136.09)

10.27 g229.25 mg
136.09 mg

Adjust pH 7.5 with 1 M KOH
Percoll solution (100 ml):
50% (v/v) Percoll
0.3 M sucrose (mw 342.30)
10 mM TES (mw 229.25)
1 mM EDTA-2Na·2H2O (mw 372.24)
10 mM KH2PO4 (mw 136.09)
1 mM glycine (mw 75.07)

50 ml
10.27 g229.25 mg
37.22 mg136.09 mg
7.51 mgAdjust pH 7.5 with 1 M KOH
Reference:Keech, O., Dizengremel, P. and Gardestrom P. (2005) Preparation of leaf
mitochondria from Arabidopsis thaliana. Physiol Plant 124: 403-409.

Correspondence: Ikuo Nishida, nishida@molbiol.saitama-u.ac.jp