laboratorycellfrac-sds-page - Laboratory 6 Week 1 Cell...

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Laboratory 6 Week 1: Cell Fractionation Introduction: Eukaryotic cells are complex and contain many kinds of membrane organelles. For instance, they contain nuclei, mitochondria, vacuoles etc. Two methods exist to study the organelles in more detail. The first is by using a variety of techniques to visualize the nuclei while still inside the cells by microscopy (i.e. cell staining and immunofluorescence). The second method involves suspending the cells in solution, and breaking them open (lysing the cells). Then the various organelles are then separated from each other by centrifugation, which then allows them to be used for further study. It is this second method that we will use in this laboratory. A procedure called cell fractionation is used to break open the cells and separate the various organelles. To perform cell fractionation, we first will suspend our cells in solution, and then we break open the cells, or lyse them. This will release the organelles inside into solution. Next, we can separate the organelles by centrifuging our solution. By using centrifugation, we can easily separate the various organelles, since the various organelles are of different mass, and density (for instance, nuclei are significantly heavier than mitochondria etc.). During centrifugation, different organelles will pellet at the bottom at specific speeds based on the mass and densities of the organelles. For instance, the heavier (larger) the organelles, the less velocity is needed to pellet the organelle. The lighter (smaller) the organelle, centrifugation must occur at a greater velocity to pellet the organelle. Therefore, if we want to separate nuclei from mitochondria, we will centrifuge at low speed. At low speed, the nuclei will pellet at the bottom, while the mitochondria will stay suspended in the solution. The left over solution after centrifugation is called the supernatant, and will contain lighter organelles (i.e. mitochondria and dissolved proteins). If we then want to separate the mitochondria from the rest of the supernatant, we can centrifuge at the appropriate speed that would pellet the mitochondria, and then remove the resulting supernatant. If we first centrifuged or cell suspension at the speed appropriate to pellet mitochondria, we would bring the mitochondria to the bottom of the tube. However, we would pellet everything that is heavier than the mitochondria (i.e. nuclei etc.). Therefore, in order to get nuclei separated from mitochondria, we must centrifuge at the lower speed first to obtain the nuclei, and centrifuge the supernatant at the higher speed to collect the mitochondria. This type of separation protocol is called differential centrifugation. In this procedure, it is possible to separate the organelles to purification because objects a similar size to our desired organelles will also pellet at the same speeds. However, these objects are significantly less concentrated in our suspension. Therefore, our pellets will are enriched for the organelle we are attempting to isolate.
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