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Chicken Liver EXPT Draft
University: University of Delhi
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![](https://d3tvd1u91rr79.cloudfront.net/140052969bcabebf660d1deee2cd81f2/html/bg1.png?Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cHM6XC9cL2QzdHZkMXU5MXJyNzkuY2xvdWRmcm9udC5uZXRcLzE0MDA1Mjk2OWJjYWJlYmY2NjBkMWRlZWUyY2Q4MWYyXC9odG1sXC8qLnBuZyIsIkNvbmRpdGlvbiI6eyJEYXRlTGVzc1RoYW4iOnsiQVdTOkVwb2NoVGltZSI6MTcyNTkwMjk0M319fV19&Signature=dxz3nqIPGYcCJWK6pCPtXUyWvS0B~loEn50~oZ2yXwXUFzPOfUSainjtQxzMhJDDuuZ-llB0WBvuCwL5-JW~pOwRzFWAg07bp7~YxoaaDtUB2ZtYHppZ5JGfJF07C~RzSspr7hrjaVjXOE74nMq6BUvh65h8uXvbUbKqSMvEeVDbtkEHMpw4MUu7m0ysszFUZC9SmObJLn~ozb8vuBO2Y6jbO25vMhnVqIydSxROoyAFNyWRZwh9F5nArb6OZhPyC28agryjkwx8fguKhVn5ZrxmWL3ptUk9oIumjFeLL5Xq0VmVTfQJB9MRJmd-wMfDAaBgNunMI3I-58~bQOWQxw__&Key-Pair-Id=APKAJ535ZH3ZAIIOADHQ)
EXPERIMENT 9
AIM: Isolation of Mitochondria from the liver and assay of marker enzyme SDH.
MATERIALS REQUIRED
• Homogenizer
• Spectrophotometer
• Cuvettes
• Centrifugation tubes
• Chicken liver
Reagents:
• 0.9% saline
• 0.15M Tris-Cl Buffer (pH=7.4) with 0.25M sucrose
• 0.4 M Tris-Cl Buffer( pH=7.4 )
• 0.2M Sodium Azide in 0.4M Tris-Cl
• 0.15M sodium succinate in 0.4 M Tris- Cl
• 0.2M DCPIP
THEORY
Centrifugation is a useful means of isolating and purifying cellular components, because most
of them differ from one another significantly in size and density. Because of difference in
shape, size and density, the various organelles or other cellular structures can be separated or
fractionated. This process is known as sub-cellular fractionation.
Homogenization: It is any of several processes used to make a mixture of two mutually non-
soluble liquids the same throughout. This is achieved by turning one of the liquids into a state
consisting of extremely small particles distributed uniformly throughout the liquid. Cells
which are a part of a solid tissue such as liver or kidney will first need to be separated from
all the connections with another cell. In some cases, this can be performed by simply
chelating the environment but in most instances the cell will need to be enzymatically or
mechanically disaggregated. This often results in subtle changes to the cells .and disrupts
such cell-cell communications. The physical means of homogenization is the combined use
of mortars and pestles, blenders, compression and/or expansion.
Differential Centrifugation: According to this preparation of broken cells is poured into a
centrifuge tube and is initially centrifuged at low centrifugal force long enough to completely
sediment the largest and heaviest sub-cellular component. On centrifugation the heaviest
particles near the bottom of the tube sediment first while the longer period is required for the
same species of the particles which are in the upper region of the centrifuge tube. By the time
these get pelleted, lighter particles originally present near the base of the tube also get
sedimented within this period. In fact, the pellet obtained is enriched with heaviest particles
but is likely to contain other particles also. The lighter particles, freed from sediment and
which are towards the top of the tube, will remain in the supernatant. The supernatant
obtained is carefully decanted and it is again centrifuged at higher centrifugal force for
sedimenting the next heavier entity in the extract. This process is continued and, at each
ensuing step, the centrifugal force as well as the centrifugation time is increased to
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