Subcellular fractionation Essay

The overall goal of subcellular fractionation is to isolate different cellular components from each other, while still preserving each components function. In this experiment we will specifically be using Mung Bean seedlings to fractionate the tissues. This experiment consisted of three parts, cellular fractionation (2A), protein quantification of cellular fractions (2B), and enzyme markers to establish fractional purity (2C). The purpose of experiment 2A was essentially to divide organelles and other cellular components from each other such as the nuclei, cytosol, and mitochondria. Researchers often want to isolate these parts of cells so they can further examine the functions of each cellular component. In order to do this, the technique of cellular fractionation is applied. Fractionation breaks down cells and disrupts cellular membranes with shearing forces and osmotic shock in a process called homogenization. After homogenization, organelles are released and can now be separated by differential centrifugation. During differential centrifugation, we continuously centrifuge our samples at steadily higher speeds to be able to fractionate different parts of a cell into their individual components. The cellular components will then pellet to the bottom based on their size and density. A mathematical formula known as Stokes Law is used to describe this separation of particles
V = 2cr2 (d-d0 / 9)
Four specimens are collected during the first lab period. These consist of homogenate, nucleic, mitochondrial, and cytosolic portions. These portions are used during the second and third lab periods.
In the second experiment, the purpose was to determine the protein content of the cellular fractions that we had collected in part A. This was done by using the Coomassie Blue method. Quantifying this protein content can help scientist later research biochemical activity of the cells. In this method, the Coomassie Blue binds to proteins to generate a blue color. Using the protein content of each cellular fraction, a standard curve was constructed to show the differences in absorbance. Making serial dilutions of Bovine Serum Albumen (BSA) allowed for the construction of this standard curve. BSA will dissolve in water to form a colorless solution. The dissolved BSA will react with Coomassie Blue to turn solutions into various shades of blue depending on the concentration of protein present in each sample. The higher the protein concentration, the deeper blue the solution will be.
During the last lab session, the enzymatic activity of the samples is determined. Enzymes can act as catalyst to speed up biochemical activities, and are used to lower the activation energy so that the reaction can successfully proceed. This analysis of enzymatic activity can tell scientists how accurate the separation of components was during homogenization. An artificial electron acceptor, 2-6-dichlorophenol indophenol (DCPIP) was used to measure the rate of the enzyme-catalyzed reaction. The reason we use DCPIP to measure the rate of the enzyme catalyzed reaction is because it is able to show a decrease in absorption before its reduction at 595nm. The cellular fractionation absorbency was then measured at different times to show if their absorbency was increasing or decreasing.