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Experiment 1 Subcellular Components PDF

This document outlines steps to isolate organelles from minced chicken liver through differential centrifugation and then analyze the biochemical components of the fractions obtained. The liver homogenate is centrifuged at increasing speeds to separate into a nuclear fraction, mitochondrial fraction, and microsomal fraction. A series of biochemical tests are then used to identify proteins, carbohydrates, lipids, and nucleic acids in each fraction based on color reactions. The tests indicate the nuclear fraction is rich in proteins and nucleic acids, the mitochondrial fraction contains carbohydrates, and the microsomal fraction is lipid-rich.

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469 views2 pages

Experiment 1 Subcellular Components PDF

This document outlines steps to isolate organelles from minced chicken liver through differential centrifugation and then analyze the biochemical components of the fractions obtained. The liver homogenate is centrifuged at increasing speeds to separate into a nuclear fraction, mitochondrial fraction, and microsomal fraction. A series of biochemical tests are then used to identify proteins, carbohydrates, lipids, and nucleic acids in each fraction based on color reactions. The tests indicate the nuclear fraction is rich in proteins and nucleic acids, the mitochondrial fraction contains carbohydrates, and the microsomal fraction is lipid-rich.

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mk
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 Isolation of organelles in their pure ANALYSIS:

form and analysis of


1. BIURET TEST
macromolecular/biomolecular
- Test for a dipeptide (protein)
components of the organelles.
- Produce pink color from dipeptide to
polypeptide.
PRINCIPLE INVOLVED: Differential
- CuSO4 – Oxidizing Agent ; NaOH – rxn
Centrifugation
medium

STEPS:

1. Extraction
- Process of isolating the organelles.
- Use Saturated Sucrose Solution
(C12H22O11)
- Under mild condition of pH and 2. MOLISCH TEST
temperature under harsh conditions. - General test for carbohydrates.
(T=4°C and <) - conc. H2SO4 – dehydrates a sugar
yielding a furfural.
2. Homogenization – Disrupting the cells - ɑ naphtol in alcohol reacts with
under a mild condition using an furfural to complete a violet/purple
osterizer. colored complex.
3. Centrifugation – Subfractionalization of
the centrifugate.

CENTRIFUGATE/RESIDUE:
I – Nuclear Fraction (Nucleic Acids)
II – Mitochondrial Fraction (Carbohydrates)
III – Microsomal Fraction (Lipids) 3. 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
4. DISCHE REACTION 5. ORCINOL/BIAL TEST
- For deoxypentose - For pentose
- Diphenylamine (DPA) – Putent - conc. HCl – Dehydrates the pentose to
carcinogen. furfural
- Orcinol Rgt. – Gives color reaction with
furfural.

Minced Chicken Liver + 20mL NSS (Normal Saline Solution) [Put to 5 vacuotainers]
1 Residue (Labeled #1) ;
Filtrate 2
Residue (Labeled #2) ;
Filtrate
3
Residue (Labeled #3)
Filtrate (Discarded)

#1: Centrifugated at speed 2 for 10 minutes.


#2: Centrifugated at speed 3 for 15 minutes.
#3. Centrifugated at speed 4 for 20 minutes.

TESTS:

MOLISCH TEST ORCINOL TEST SUDAN IV DPA BIURET


PRECIPITATE (5 Drops) (2 Drops; Water (w/ 2 Drops (w/ 1 Drop conc. ( 2 Drops CuSO4
bath for 5 mins. CHCl3/CH2Cl2) H2SO4 ; Water bath and NaOH)
for 5 mins)
I +++ + + +++ +++
2 ++ ++ ++ ++ +
3 + +++ +++ + ++

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