Question

Describe a protocol for isolating mitochondria from rat liver and for estimating the purity of the preparation.

Answer 1

Preparation of isolation buffer:

Isolation buffer consists of:

40.99 grams of 225mM Mannitol + 35.67 grams 75mM sucrose + 0.0744 grams of 0.2mM EDTA being dissolved in 1000 ml of water.

Isolation protocol:

1. Sacrifice the rat and dissect the liver out and put it in isolation medium (ice-cold).

2. Use 1.5 g of the tissue for the experiment.

3. Cut the tissue into small pieces and add the isolation medium in drops while cutting.

4. Homogenize the tissue at 1,000 rpm followed by the addition of more medium.

5. Transfer the content to the 50 ml Falcon tube and make up the volume to less 10% homogenate i.e 1 g tissue : 15 – 20 ml homogenate.

6. Centrifuge at 1,000 rpm for 10 min at 4 °C.

7. Filter the supernatant and transfer it into another tube and again centrifuge at 6,200 rpm for 10 min at 4 °C.

8. Discard the supernatant and resuspend the sediment of mitochondria in 15-20 ml of isolation medium/gram of tissue.

9. Centrifuge at 6,200 rpm for 10 min at 4 °C.

10. Discard the supernatant and re-suspend mitochondrial sediment and transfer it into fresh tubes and store at -20 °C.

Estimating the purity of the preparation:

Western blotting can be used to check the markers of mitochondria.

Markers of mitochondria:

Voltage dependent anion channel (VDAC/porin) ----> an outer mitochondrial membrane marker

HSP-60 ---> soluble protein in mitochondrial inner matrix space

Prohibitin ---->  inner mitochondrial membrane marker

The presence of all three markers ensure that the isolated mitochondria samples are intact and agree with the functional studies.

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