To Understand the Principles of Differential Centrifugation - Medical Science Assignment Help

Assignment Task:

Task:

LEARNING OBJECTIVES:

1. To understand the principles of differential centrifugation.

2. To be familiar with the use of marker enzyme assays for determining cell fraction purity and enzyme activity.

Aims of experiment:

To separate organelles from spinach leaves using differential centrifugation and use a marker enzyme assay to confirm isolation of mitochondria. Introduction - Cell fractionation using homogenisation & centrifugation Eukaryotic cells are complex and contain several different membrane-bound organelles that perform a variety of specific functions, for example lysosomes containing digestive enzymes to breakdown molecules in the cells, mitochondria to synthesize ATP, endoplasmic reticulum which is a site of protein synthesis. Since cells are so diverse in their structure and function, the types and numbers of organelles varies hugely in different cell types, from cells with no organelles eg. mammalian red blood cells, to cells with many different organelles eg. liver cells and epithelial cells lining the gastrointestinal tract.

Several methods are available for isolating subcellular organelles, and one of the most common is centrifugation. To separate organelles via centrifugation it is important to first homogenise the cells/tissues to break open the cell membrane (and cell wall if present). This releases the organelles. Other methods of homogenisation include filtration through small pores in a filter, grinding of cells with glass beads or using a mortar and pestle, sonication of cells with ultrasonic vibrations and solubilisation of cell membranes using detergents. The method used depends on the type of tissue and the aim of the experiment. In this laboratory you will use a mortar and pestle to homogenise your cells. During homogenisation isotonic sucrose is usually added to the homogenisation buffer to prevent osmotic rupture of organelle membranes. Two types of centrifugation procedures are commonly used for the separation of sub-cellular organelles, namely differential centrifugation and density gradient centrifugation.

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