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THE PRACTICAL REPORT
Student Name-
University-
PRACTICAL 2:
Isolation of subcellular fractions of Mitochondria from mouse liver cells & BCA Essay
AIM- To isolate mitochondria from liver cells and to carry out BCA essay
Introduction:
The powerhouse of cell, mitochondria, is an essential cell organelle of the cell that comprises of
important enzymes responsible for chemical reactions of cellular respiration. Many research
studies have been conducted to understand the structure and function of mitochondria in various
neurodevelopmental disorders such as Autism Spectrum Disorders or many types of cancers
(Siddiqui, Elwell & Johnson, 2016).
To study about the functions of mitochondria in liver cells isolation of subcellular fractions is
done through differential centrifugation technique. Successful isolation and purified fractionation
of mitochondria allows characterized study of protein and its components. Subcellular
fractionation is used to prepare samples for further analysis of mitochondria.
Differential centrifugation is a technique used to prepare a crude extract of mitochondria and
purify it using a density gradient. A crude extract is prepared using this protocol within an hour.
The subcellular fraction of mitochondria is used further in enzymatic assays such as BCA to
obtain total protein concentration in mitochondria and electrophoretic analysis.
Mitochondria comprise soluble proteins and membrane proteins. Altered protein concentration in
mitochondria leads to several fatal disorders. BCA is a protein assay which is used to determine
the total concentration of proteins in subcellular fractions of mitochondria on the basis of a
standard curve. The assay works on the principle of reduction of cupric oxide to cuprous oxide
by protein in a biuret reaction resulting in the formation of bicinchoninic acid (purple color).
Materials and Methods:
Subcellular fractionation of mitochondria from mouse liver cells-
Fig. 1.1: Differential centrifugation
1. 2 mm freshly dissected pieces of liver were kept over ice.
2. Tissue sample around 500-600 mg was transferred in cryovials (1 ml) and sterilized
storage solution prepared by adding 210 mM mannitol and 70 mM sucrose in 20%
DMSO was added.
3. Tissue sample was then stored in liquid nitrogen.
4. -
C
0.4% BSA in 100 mM Tris-HCl of pH 7.5.
5. 1-2 mm pieces of the sample tissue were sliced with the help of a sterile scalpel.
6. The diced sample was then transferred to a centrifuge tube.
7. 8 mL pre-chilled isolation buffer and protease inhibitors was prepared with 250 mM
sucrose and 0.2mM 2NaEDTA dissolved in 10 mM Tris - HCl of pH range from 7.5 -
8.0.
8. IKA tissue grinder was used to homogenised tissue at a speed of 3 x 7 sec bursts using
ice.
9. Homogenised tissue sample was divided into five 2 mL microcentrifuge tubes and
labelled as:
. L CL tube and kept on ice.
. C f f ˚C v f
prepared homogenate.
12. The supernatant was transferred to Mito tube containing 1 mL filter tip.
13. Supernatant was centrifuged aga f ˚C f SN
and stored on ice.
. T f L f -buffer to
remove clumps of cells.
15. Again the tube was centrifuged at 12,000 x g for 10 min ˚C
pipetted out and kept in SN2 tube.
6. T L f -buffer and mixed well.
17. Mitochondria was pelleted in a cooling centrifuge at 12,000 x g for 10 min.
18. The supernatant was pipetted off again and kept in SN3 tube.
9. L f -buffer was added to resuspended mitochondrial pellet and the mixture
was kept on ice.
BCA Protein Assay-
The standard curve was prepared on...