Chem 5a Biochemistry

Alyzza Rose Manayon

Phoebe Mhay Porras
Expt. 1  The Living Cell
I. Objectives:
II. Results and Observation:
A. Separation of Cell Organelles:

Process Observation:
Preparation of  In washing the freshly excised liver, the blood must be removed using
Tissue ice cold water. After removing the blood, blot with a filter paper. The
liver must be minced finely using a knife or a scissor. Weigh the
minced liver in a beaker. Add 5ml of Homogenizing Medium in the
beaker that contains the minced liver. Homogenize using a
homogenizer at a low speed for 5-10mins. Transfer the homogenate
in a centrifuge tubes for centrifugation
B. Differential The homogenate is centrifuged at 600 rpm for 10 minutes forming
Centrifugation sediment 1 and supernate 1. Supernate 1 is centrifuged at 8,000 to
15,000 rpm for 10 minutes where the nucleus is settled at sediment 1.
(describe the Sediment 2 and supernate 2 are formed which are then centrifuged at
different 15,000 rpm for 10 minutes where mitochondria is settled at sediment
fractions) 2. Sediment 3 and supernate 3 are formed. Lysosomes are settled at
sediment 3 and microsomes, soluble proteins, enzymes, and
inorganic ions are settled at supernate 3.
 

B. Qualitative Analysis of the Cell Fraction: Write only +/- on the designated space for
present/absent. Compare relative amounts by adding more + if the color of the result is
darker:

Sample 1 Sample 2 Sample 3 Sample 4

(Sediment 1) (Sediment 2) (Sediment 3) (Supernate 3)
1. Carbohydrates
(Molisch Test-Violet ring) +++ ++ + ++++

2. Proteins
(Biuret Test-violet + ++ +++ ++++
solution)
3. Lipids
(Sudan IV  red-orange + ++++ ++ +++
layer)

4. Nucleic Acids
  a.) DNA
(Dische/Feulgen’s +++ + ++++ ++
grayish - blue solution)

  b.) RNA
(Orcinol green complex) ++++ + ++ +++

III. Discussion:
Guide Questions:
1.    Why perform differential centrifugation?
 
Differential centrifugation must be performed to isolate different heterogeneous
solutions and to enrich cell organelles. It also accelerates the separation process.
 
2.    What are the organelles found in the different fractions?
The organelles found first are in Sediment 1. Sediment 1 is composed of the Nucleus
and the Unbroken cells. Next, sediment 2 is composed of the mitochondria. Lastly, The
organelle found in Sediment 3 is Lysosome while Supernate 3 is composed of
microsomes, soluble proteins, enzymes, and inorganic ions.
 
 
3.    What are the components of these fractions 
After the initial/first centrifugation, sediment 1 which is the larger particles will settle. In
the second centrifugation, the second-largest particle which is sediment 2 will settle.
Lastly, after the third centrifugation, sediment 3 and supernate 3 which is composed of
the liquid portion composed of suspended particles will settle.
                                                                                                                                            
                 
IV. Conclusion:

Therefore we conclude that the importance of differential configuration is not only

evident as it accelerates the separation process, it also separates heterogeneous
solutions and determines the different organelles present in each fraction. Within the
three configurations, Nucleus, Mitochondria, Lysosome, microsomes, soluble proteins,
enzymes, and inorganic ions are organelles that are present. In each tests, we were
able to identify the amount of biomolecules in each samples. All samples contained all
the biomolecules which we identified by their colors. The amount of biomolecules in
each samples were identified based on the intensity of their colors.