Cell Fractionation by Centrifugation

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Blanca Mbuto 02/19/2012 Biochemistry Lab 4422 Cell Fractionation by Centrifugation Abstract This experiment was performed to investigate organelles, membranes, cellular components, and enzymes. Our concentration was to observe and determine if the chlorophyll contained broken chloroplasts or intact chloroplasts extracted from spinach leaves. A percoll density gradient was prepared followed by the separation of organelles. Chlorophyll was analyzed by using a spectrophotometer. We compared the absorbance readings of each tube and selected three (3) tubes to be analyzed. A drop of sample was placed on a slide and mounted on a microscope for chloroplast examination. We concluded that broken chloroplasts have rougher edges and are dark green in color. Their glow under the microscope is not as significant as the intact chloroplasts which appear to have a yellow halo surrounding them and their edges are significantly smoother. Introduction Although biochemical analysis requires disruption of the anatomy of the cell, gentle fractionation techniques have been devised to separate the various cell components while preserving their individual functions. Cells can be broken up in various ways. They can be subjected to osmotic shock or ultrasonic vibration, forced through a small orifice, or ground up in a blender. These procedures break many of the membranes of the cell (including the plasma membrane and membranes of the endoplasmic reticulum) into fragments that immediately reseal to form small closed vesicles. Materials and Methods This experiment was conducted according to the Cell Fractionation by Centrifugation Protocol (Stephens 2012). Results According to the data collected for all nine (9) samples we determined that three (3) samples should be examined under the microscope. Sample one (1), eight (8), and nine (9) we chosen to be analyzed due to their

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