Extraction of Glycogen 125122221121.pptx

1. Extraction of Glycogen from
Chicken Liver
Dr .Waseem Safdar (Assistant Professor) NDBS NUMS
Biochemistry-II
(Practical-2)

2. Content
Introduction & Principle
Materials
Method
Result & Interpretation
Conclusion
Dr .Waseem Safdar Biochemistry-II (MIC-635) NDBS - NUMS

3. Introduction
Glycogen is a branched polysaccharide composed of α
-
D-glucose residues
joined by glycosidic bonds.
Glucose residues joined by the α
-
1,4 bonds, and branches are formed by α
-
1,6- glycosidic bonds..
High degree of glycogen chain branching helps to improves the solubility
Glycogen is the major glucose storage polymer in animals.
The compactness of these polymer chains allows large amounts of carbon
energy to be stored in a small volume.
Dr .Waseem Safdar Biochemistry-II (MIC-635) NDBS - NUMS

4. Principle
The concentration of glycogen is higher in the liver (10% by weight) than in
muscle (2% by weight).
The carbohydrate tests can be divided into two classifications based on the
mechanism of action .
The first test involves the use of dehydrating acids followed by
condensation of reagents.
The Molisch’s Test should show positive results for all carbohydrates.
Glycogen can be separated from other proteins by mincing, grinding, and
boiling the liver
Dr .Waseem Safdar Biochemistry-II (MIC-635) NDBS - NUMS

5. Materials
 0.1% Methanol
 95% ethanol
12M H2SO4
 Molisch’s Reagent
 Chicken liver
 50 mL beaker
 100 mL beaker
 glass Stirring rod
 3 Test tubes
Dr .Waseem Safdar Biochemistry-II (MIC-635) NDBS - NUMS
 Wire Gauze
 Scissor
 25 mL Graduated cylinder
 Water bath
 Test tube rack
 Test tube holder
 Hot plate
 Bunsen Burner
 Petri plates
 Mortar and Pestl
 0.01M Iodine

6. Methods
• Wash chicken liver with distilled water in a Petri plate
• Take 20 grams of washed chicken liver in mortar and pestle
• Add 50 mL of phosphate buffer (7.4 pH)
• Mince the sample into a homogenize sample to even and smooth
consistency and then transfer to falcon tubes 25 mL each.
• centrifuge at 3000 rpm for 10 minutes
• Collect supernatant in a test tube
• Add 1 mL of 10% acetic acid
Dr .Waseem Safdar Biochemistry-II (MIC-635) NDBS - NUMS

7. Methods
• Boil the sample in water bath for five minutes
• Let it cool down and then centrifuge at 3000 rpm for five minutes
• Collect supernatant to a new test tube and place it in a refrigerator for 30
min
• Add 5 mL of Absolute ethanol and place test tube in refrigerator for another
60 min
• Centrifuge at 3000 rpm for 10 minutes
• Discard supernatant and Collect precipitate (Glycogen)
• Dissolve the precipitate in 5 mL distilled water.
Dr .Waseem Safdar Biochemistry-II (MIC-635) NDBS - NUMS

8. Methods
Hydrolysis
• Take 3 mL of Glycogen solution and add equal amount of 6M HCl.
• Boil the test tube in a boiling water bath for 30 minutes.
• Add 3 mL of concentrated ammonium hydroxide
Molisch test
• Take 10 drops of hydrolyzed sample and add 10 drops of Molisch reagent
• Mix thoroughly
• TILT the test tube carefully and add one mL of H2SO4 slide down the side of
the test tube to form a layer at the bottom and observe the color
Dr .Waseem Safdar Biochemistry-II (MIC-635) NDBS - NUMS

9. Results & interpretation
Description Result
Chicken liver Fresh, radish
Homogenized mixture Light yellow, stinky odor
Precipitation Stinky, white to light brown
After refrigeration White precipitates
Molisch test Hot and Purple ring formation
Iodine test reddish-brown or blue-black
color
Dr .Waseem Safdar Biochemistry-II (MIC-635) NDBS - NUMS

10. Conclusion
Glycogen will give a positive result in molisch’s reagent and Iodine solution
because it is a carbohydrate.
Dr .Waseem Safdar Biochemistry-II (MIC-635) NDBS - NUMS

11. THANKS
Dr .Waseem Safdar Biochemistry-II (MIC-635) NDBS - NUMS