Antibodies Plant/Algal / Environmental Stress / Plant Oxidative Stress

Agrisera Western Blot protocol and video tutorials

Protocols to work with plant and algal protein extracts

Plant Mitochondria Preparation

In this example of mitochondrial preparation used material are etiolated corn seedlings, however, it can also be applied to prepare crude mitochondria from any plant tissue. The volume has to be adjusted, to match the amount of plant material available. For some plant tissues, it can be useful to include sand when grinding the tissue.

Important remarks before you start:

• 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 pipeting 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 - 35 ml centrifuge tubes
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 hours. Plant material should not contain any harder plant parts. Proposed tissue amount 15 grams/60 ml of homogenization buffer. Leaf tissue should be quickly and thoroughly cut followed by 3 minute of homogenization.

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

Procedure:
1. Place ½ 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 prearation.

Solutions Required

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

1 Liter
Mannitol 63.8 g
Mops 6.25 g
1 mM EDTA 2 ml of 0.5 M stock

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

250 mls
Mannitol 13.7 g
Mops 1.05 g
1 mM EDTA 0.5 ml of 0.5 M stock

SUCROSE CUSHION: 0.6 M Sucrose

100 mls
Sucrose 20.5 g

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

100 ml
Sucrose 8.56 g
Mops 0.628 g

Courtesy Dr. Thomas Elthon