Isolation Techniques for Cell Components

Isolation techniques for Cell Components

Objective: To study isolation techniques, staining and microscopic .detection of cell organelles and other components

(1) Isolation of Glycogen Granules Glycogen is a colorless solid material that can be dissolved in cold water to form a viscous solution . Glycogen gives red color with iodine. Material: - Rat that has been fed with starchy food - Mortar and pestle apparatus - 10 % trichloroacetic acid - Centrifuge and centrifuge tubes - Alcohol and iodine. steps: 1- Dissect rat and take its liver, cut it to small pieces. In a mortar crush liver with grated ice and cooled washed water with cold trichloroacetic acid (10%) (1 ml of acid for each 1 gram of liver). 2- Put mixture in glass beaker, then distribute it into centrifuge tubes. 3- Put centrifuge tubes into the centrifuge at 2000 rpm for 5 min. 4- Take the upper supernatant containing glycogen into a 200 ml flask, and discard the pellet. (To insure obtaining maximum amount of glycogen , wash the mortar with cold solution of trichloroacetic acid and centrifuge again). 5- Take the upper supernatant into flask , and discard pellet. 6- Add 95% alcohol to the supernatant (where amount of alcohol is twice amount of liver extract), shake flask well and leave it aside. Observe formation of precipitant. We can accelerate formation of precipitant by adding salt and warming the mixture. 7- Discard supernatant, and collect the white precipitant (glycogen). We can further purify the glycogen by rewash it with water and precipitate with alcohol. 8- Apply small amount of glycogen into a clean glass slide, then add a drop of dil. iodine solution. Examine slide in microscope.

Draw Slide: (2) Isolation of Mitochondria from Liver

Objective: Separate cellular organelles, then isolate and detect mitochondria

To study cell’s organelles and other components, cytoplasmic membrane should be destroyed (lysed) with conserving components structure. The .result from lysis is called cell extract

:In the experiment, liver extract will be centrifuged 3 times First centrifuge: nuclei and other heavy cellular parts will precipitate, and .mitochondria will be in the supernatant .Second: Mitochondria precipitate in pellet Third: Mitochondria separate from other components as proteins, and .mitochondria will precipitate in pellet as final result

Steps: 1- Cut a 2 gm piece of liver, put it in mortar with 18 ml of homogenizing buffer 2- Grind mixture with electric homogenizer for 2 minutes 3- Put 5 ml of homologous mixture in 15 ml centrifuge tube 4- Put centrifuge tubes in centrifuge (in balance, i.e. even number of tubes of same liquid size and each two tubes opposite to each other), centrifuge at 2400 rpm for 10 min. 5- Transfer supernatant in new centrifuge tube (and discard pellet) 6- Centrifuge for maximum speed (6000 rpm) for 10 min 7- Discard supernatant, and add 5 ml of suspension buffer on pellet, and mix with dropper to make homologous mixture 8- Centrifuge at maximum speed for 10 min. 9- Discard supernatant, add 40 ml of suspension beffer 10 – In new test tube mix 6 drops of Jenus green B stain + 200 μl of mitochondria suspension , and observe color of suspension. 11- Apply a drop of suspension on a slide, cover with cover slide and examine with microscope high power. `