Cell Fractionation
Cell Fractionation
i) EQUIPMENT:
Cold Liver, Blender, Gauze, Fridge, Isotonic Solution, Centrifuge, Tubes, Scalpel,
HOMOGENIZATION
• Take chilled liver tissue and cut into smaller pieces.
• Place liver into blender with cold isotonic solution.
• Blend until you have a thick soup like substance (homogenate).
• Filter the mixture to remove large debris through gauze into a tube.
DIFFERENTIAL CENTRIFUGATION
• Attach tubes of homogenate to the refrigerated Centrifuge and turn on to spin the tubes and separate the organelles in order of mass. As per fig 1.
• Centrifuge @ 600 x g for 5 minutes to separate Nuclei into a pellet at the bottom of the tube A.
• Pour the remaining Supernatant fluid above the pellet from tube A into tube B and place in the centrifuge for the next spin cycle.
• Centrifuge @ 3000 x g for 20 minutes to separate the Mitochondria and form a pellet in the bottom of tube B.
• Pour the remaining Supernatant fluid from tube B into another tube and re-suspend Tube B’s pellet by adding isotonic solution. And store at -4c.
Fig1 http://site.motifolio.com/images/Differential-centrifugation-6111181.png ii) STUDY OF MITOCHONDRIA FUNCTION
Too ensure that the study is correct the Mitochondrial Pellet will be cut up and the exact amounts of pellet and isolate solution will be placed in the tubes. Also the tests will be repeated to see if the results are the same.
1. This study will be to find out if chemical reactions of the Mitochondria are sped up or slowed down at various temperatures.
9 tubes will be heated at 5 degree Celsius differences .e.g. 0, 5, 10, and 15 up to 40 degrees Celsius. When the tube has reached its required temperature the isotonic fluid is analyzed to see if there are any changes such as; Amount of fluid( increased or decreased), any new chemicals?
2. This will involve adding an amount of sugar to the Mitochondria to see if it is taken in and used, similarly to the process of cellular respiration. The
i) EQUIPMENT:
Cold Liver, Blender, Gauze, Fridge, Isotonic Solution, Centrifuge, Tubes, Scalpel,
HOMOGENIZATION
• Take chilled liver tissue and cut into smaller pieces.
• Place liver into blender with cold isotonic solution.
• Blend until you have a thick soup like substance (homogenate).
• Filter the mixture to remove large debris through gauze into a tube.
DIFFERENTIAL CENTRIFUGATION
• Attach tubes of homogenate to the refrigerated Centrifuge and turn on to spin the tubes and separate the organelles in order of mass. As per fig 1.
• Centrifuge @ 600 x g for 5 minutes to separate Nuclei into a pellet at the bottom of the tube A.
• Pour the remaining Supernatant fluid above the pellet from tube A into tube B and place in the centrifuge for the next spin cycle.
• Centrifuge @ 3000 x g for 20 minutes to separate the Mitochondria and form a pellet in the bottom of tube B.
• Pour the remaining Supernatant fluid from tube B into another tube and re-suspend Tube B’s pellet by adding isotonic solution. And store at -4c.
Fig1 http://site.motifolio.com/images/Differential-centrifugation-6111181.png ii) STUDY OF MITOCHONDRIA FUNCTION
Too ensure that the study is correct the Mitochondrial Pellet will be cut up and the exact amounts of pellet and isolate solution will be placed in the tubes. Also the tests will be repeated to see if the results are the same.
1. This study will be to find out if chemical reactions of the Mitochondria are sped up or slowed down at various temperatures.
9 tubes will be heated at 5 degree Celsius differences .e.g. 0, 5, 10, and 15 up to 40 degrees Celsius. When the tube has reached its required temperature the isotonic fluid is analyzed to see if there are any changes such as; Amount of fluid( increased or decreased), any new chemicals?
2. This will involve adding an amount of sugar to the Mitochondria to see if it is taken in and used, similarly to the process of cellular respiration. The
Bibliography: Boyle M and Senior K. (2008) Human Biology. 3rd ed. London: Collins. Parker J and Honeysett I. (1998) Revise AS Biology. London: Letts and Lonsdale. http://biology.about.com/od/cellularprocesses/a/cellrespiration.htm http://site.motifolio.com/images/Differential-centrifugation-6111181.png