Objectives

to learn the principles of separation of subcellular components.

identification of the principles in using differential centrifugation for isolating
subcellular components.
identification of the biomolecules present in the different fractions using qualitative
tests.
composed of cell organelles that have different biomolecular compositions.
"building blocks of life"
The Cell
The Experiment
The specific technique that was employed in this experiment is differential
centrifugation.
Tissue homogenization is a process that involves the breaking apart of cells.
Releasing the organelles and cytoplasm.
Differential Centrifugation
is done by putting the solution of homogenized organelles in a variable speed
centrifuge and rotating them at a higher rate of speed.The stepwise increase in the
speed of centrifugation is highly essential.
More dense particles settle at the bottom and become separated from less dense
particles.
Successive speed of centrifugation is increased until the target particle has settles
at the bottom.
Steps
EXTRACTION
- Process of isolating the organelles.
- Use Saturated Sucrose Solution (C12H22O11)
- Under mild condition of pH and temperature under harsh conditions (T=4C and
<).
HOMOGENIZATION
- Disrupting the cells under a mild condition using an osterizer
CENTRIFUGATION
- Sub-fractionalization of the centrifugate
Repeated centrifugation at progressively higher speeds will fractionate
homogenates of cells into their components. In general, the smaller the subcellular
component, the greater is the centrifugal force required to sediment it
Reagents
BIURET TEST
- Test for a dipeptide (protein)
- Produce purple color from dipeptide to polypeptide.
- CuSO4 (Oxidizing Agent); NaOH (reaction medium)
MOLISCH TEST
- General test for carbohydrates.
- conc. H2SO4
- Dehydrates a sugar yielding a furfural.

- -naphtol in alcohol reacts with furfural to complete a violet/purple colored

complex.
SUDAN IV
- Classified as oxidizing dye, carcinogen.
- Hydrophobic dye that dissolves on hydrophobic solvents, lipids.
* Non-specific test because all hydrophobic will dissolve in Sudan IV.

Centrifugate/ Residue
I - Nuclear Fraction (Nucleic Acids)
- An operational term for the material deposited by low-speed centrifugation of a
homogenate of eukaryotic cells or tissue.
- It consists predominantly of nuclei together with unbroken cells.
II - Mitochondrial Fraction (Carbohydrates)
- An operational term for the more rapidly sedimenting components (for example 10
000 15 000 g for 30 min) of the cytoplasmic fraction of a homogenate of
eukaryotic cells or tissue.
- It generally consists predominantly of mitochondria but may also contain varying
numbers of secretion granules, lysosomes, microbodies, or other intracellular
organelles.
III - Microsomal Fraction (Lipids)
- An operational term for the subcellular fraction of a homogenate of a eukaryotic
cell or tissue that, on differential centrifugation, is sedimented at 105 g
- The fraction from some tissues, such as liver, although consisting largely of
microsomes, commonly also contains free ribosomes and fragments of the plasma
membrane, of the Golgi apparatus, of mitochondria, and of other subcellular
structures.
Discussion
A multi-step process which puts the homogenized chicken liver under different
speeds in every steps.
Homogenization breaks the liver cells as to release the cell organelles ready for
centrifugation and application of different tests.
Nuclei are the heaviest subcellular component.
The next is composed of the mitochondria
The last components were the lighter cell organelles such as golgi bodies,
ribosomes, vesicles and cell membrane.
Nuclear DNA, or nuclear deoxyribonucleic acid (nDNA), is DNA contained within a
nucleus of eukaryotic organisms. In mammals and vertebrates, nuclear DNA
encodes more of the genome than the mitochondrial DNA & ribosomal RNA. It
composed of information inherited from two parents, one male, and one female,

rather than matrilineally. Of the 23 pairs of chromosomes in the human body,

Results
DNA
Ribosomal ribonucleic acid (rRNA) is the RNA component of the ribosome, and is
essential for protein synthesis in all living organisms. It composes the predominant
material within the ribosome. Mitochondria & Nuclear has a small number of RNAs.
RNA
From the table one can conclude which biomolecule is abundant in the cell, in this
case RNA and Protein are.The experiment is limited to two sediments and
supernatants because of limited capabilities of the centrifuge.
Conclusion
Being the densest constituent, the nucleus is thirst to settle at the bottom. it is then
followed by the precipitation of the mitochondria and lastly the supernate is
composed of the least dense components such a golgi apparatus, ER, vesicles,
ribosomal units ant cell membrane.
The nucleus gave a positive result for the biuret test which indicates the presence of
proteins, A positive result for Sudan IV test indicating the presence of lipids .
The mitochondria contain the 4 biomolecules carbohydrates, proteins, lipids and
nucleic acid.
The supernate gave a positive result for the biuret test , molisch test and sudan IV
indicating the presence of carbohydrates, proteins and lipids.