Protocols > Mitochondrial Fraction

This mitochondrial preparation is for etiolated corn seedlings, but it can be used to prepare crude mitos from any plant tissue. Change the volumes to match the amount of plant material available. For some plant tissues, it is useful to add a little sand when grinding the tissue.

Important remarks before you start:

• The whole procedure should be done at 4ºC
• All centrifugations should be done at 4ºC
• Whole procedure should be done as fast as possible
• Plant material should be homogenized very quickly
• Very careful pipetting is recommended
• After isolation membrane integrity can be checked (should be around 80%). It can be done using Cytochrome C oxidase kit (Sigma)

Materials

• 250 ml grinding media (1.8 g PVPP and 0.34 g L-cysteine)
• 1 liter beaker and large funnel
• 2 large, wetted muslins
• 8 centrifuge tubes (35 ml each)
• large pestle and mortar
• SS-34 rotor Sorvell centrifuges, or JA-20 for Beckman
• 75 g corn shoots

Precautions

Until the time of mitochondrial isolation all plants should be kept in the darkness for min 8 h. Plant material should not contain any harder plant parts. Proposed tissue amount 15 g/60 ml of homogenization buffer. Leaf tissue should be quickly and thoroughly cut followed by 3 min of homogenization.

It is important to keep all materials cold (4°C) and work swiftly while grinding and spinning.

Procedure

1. Place half of the shoots into the mortar and add grinding media to nearly cover the shoots. Grind until shoots are unrecognizable, add additional grinding medium, and pour pulp into muslin and squeeze. Grind the remaining shoots in a similar way and use the second clean muslin to filter. Divide filtrate evenly among the 8 tubes.
2. Spin at 6500 rpm for 2 min at speed.
3. Transfer supernatant into 8 clean tubes.
4. Spin at 12,750 for 5 min at speed.
5. Discard supernatant. Place the tubes on ice at an angle with pellet-side up.
6. Wash the pellets with 1 ml of Wash Media to remove yellow slime.
7. Add 5 ml of Wash Media and resuspend the pellet with a pipetman while leaving behind the small white mass (starch). Pool into two tubes.
8. Spin at 6500 rpm for 2 min at speed.
9. Pipet supernatant into clean tubes while leaving behind slimy film.
10. Underlay the supernatant with 8 ml of Sucrose cushion.
11. Spin at 9250 rpm for 20 min at speed.
12. Aspirate supernatant.
13. Resuspend each pellet in a suspension medium of choice, this is your crude mitochondrial preparation.

Solutions Required

GRINDING MEDIUM: 350 mM Mannitol; 30 mM Mops, 1 mM EDTA, pH 7.6

Dilute in ddH2O up to a volume of 1 Liter:

WASH MEDIUM: 300 mM Mannitol, 20 mM Mops, 1 mM EDTA, pH 7.2

Dilute in ddH2O up to a volume of 250 ml:

SUCROSE CUSHION: 0.6 M Sucrose

Dilute in ddH2O up to a volume of 100 ml:

SUSPENSION MEDIUM: 250 mM Sucrose, 30 mM Mops, pH 7.2

Dilute in ddH2O up to a volume of 100 ml:

Courtesy of Dr. Thomas Elthon