lab 3 partial lab report.docx - Subcellular Distribution...

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Subcellular Distribution and Structure of Ribulose1,5-Biphosphate Carboxylase (Rubisco)Katelyn CisnerosLab Partners: Kara Lamb, Dusan Nikolic-Dorschel, Aaron PariseFebruary 25, 2019 Cell Biology PCB3023-901
Materials and MethodsHomogenizationThe first step of this experiment was performed by the TA. This step was to create a Spinach Homogenate that was ready to be centrifuged. The sample appeared dark forest green and it was aqueous. We measured 200 μLof the Homogenate and dispensed it into a microfuge test tube. The sample was labeled asCrude Homogenate (CH) and placed on ice. This homogenate on a cellular level would contain cell walls, nuclei, leaf debris, and whole cells. Next we are centrifuged the Spinach Homogenate to separate the components of the cell in 3 more different subcellular isolation fractions. The remainder of the Spinach Homogenate given to us was placed in the centrifuge for 2 minutes at 10,000 x g. After centrifugation, a pellet had formed at the bottom of the test tube and it needed to remain undisturbed until its final spin. Above the pellet was the supernatant and 200 μLwas removed and dispensed into a microfuge test tube. The sample would be labeled Low Speed Supernatant (LSS) and placed on ice. On a cellular level this is mostly chloroplast, mitochondria and lysosome sediments. The color of the original Spinach Homogenate is becoming more clear and is lighter in color similar to a lime. The remaining supernatant of the spinach homogenate was centrifuged again but this time for 10 minutes at 105,000 x g. After centrifugation, 200 μLof the supernatant was transferred into another microfuge test tube. The sample was labeled as High Speed Supernatant (HSS) andplaced on ice. The remaining supernatant was removed from the original sample ofspinach homogenate, so that only the pellet remained dry. 100 μLof homogenization buffer was added to the pellet in the microfuge test tube and vortexed. After this process, 200 μLwas removed from the sample and dispensed into a microfuge test tube. The final sample was labeled as High Speed Pellet (HSP) and placed on ice. This final fraction on a cellular level would containthe Golgi Apparatus and the Endoplasmic Reticulum membranes. Cell Fractionation Cell Fractionation is the separation of cellular organelles or components. This was the goal of the first step when making the different fractions of the Spinach Homogenate after placing it in the centrifuge 3 times performing differential centrifugation. The process increasingly sediment smaller particles. Wedisrupted the cell walls so we can see what is going on inside the cell beyond the membrane and into the organelles. The first spin contained the nucleus and intact cells. The second involved the mitochondria and lysosomes and the final spin contained the Golgi Apparatus and Endoplasmic Reticulum membranes. Each of

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