Aim:
To isolate mitochondria from the given sample
Objective:
The isolation of mitochondria is useful in order to study and explore fundamental processes including mitochondrial respiration, metabolic activity, protein import, membrane fusion, protein complex assembly, as well as interactions of mitochondria with the cytoskeleton, nuclear encoded mRNAs, and other organelles.
Principle:
The nucleus has two membranes, and mitochondria are membrane-bound organelles. It’s a fairly smooth membrane. However, the inner membrane is extremely twisted, creating cristae, or folds. The surface area of the inner membrane is significantly increased by the cristae. These cristae are where food (sugar) and oxygen combine to form ATP, the cell’s main energy source.
By using cell fractionation methods and ultracentrifugation, mitochondria can be isolated in the laboratory.
Materials Required:
 Pea seedlings
Isolation medium (pH 7.8) containing 30mM (3-(N-morpholino) propane sulfonic acid (MOPS), 0.3 M mannitol, 4mM cysteine, 1mM Ethylenediaminetetraacetic acid (EDTA) and 0.1 % (w/v) defatted BSA (pH 7.8). (For green leaf tissue, 0.6% (w/v) insoluble acid washed polyvinyl pyrrolidone and BSA concentration is increased to 0.2% (w/v).
Resuspended medium- isolation medium without cysteine
Sucrose gradient (non-linear): 1.8 M. 1.5M, 1.2M, and 0.6 M sucrose solution separately prepared in 10mM MOPS or phosphate buffer (pH 7.2, 0.1% (w/v) BSA.
Measuring cylinder
Cheese cloth, Glass funnel, Test tubes, centrifuge tube
Procedure:
- Grind 100-200gm deveined tissue in mortar and pestle with two volumes of chilled isolation medium to a paste.
- Filters homogenate through about eight layers of clean cheesecloth in a glass funnel into an iced 16 X 150mm test tube.
- Centrifuge at 700- 1000 x g for 10 minutes to remove cell debris and starch grains.
- Decant the supernatant into a clean cold centrifuge tube, discard sediment
- Centrifuge the supernatant at 1000 x g for 20 minutes and discard the supernatant.
- Resuspend pellet in ice-cold 40-50 ml of resuspended medium with a clean, ice-cold pipette.
- Centrifuge at 250 x g for 10 minutes (to reduce contaminant).
- Centrifuge the supernatant at 1000 x g for 10 minutes. Suspended the pellet in 1-2 ml of resuspension medium.
- Prepare the non- linear sucrose gradient by carefully adding 6 ml each of 1.8 M, 1.5 M, 1.2 M and 3 ml 0.6 M sucrose solution. Layer 1 ml of crude preparation onto the gradient.
- Centrifuge at 4000 x g for 45 minutes
- Collect the band at 1.5 M -1.2 M interface using a hypodermic needle. Dilute isotonic conditions (0.3M) by slowly adding buffers.
- Pellet the mitochondria by centrifugation at 1000 x g for 15 minutes and suspend in a small volume of suspension medium.
- Observe under a microscope.
Note:
Keep all the equipment and materials ice cold.