Cauliflower Cell Lab

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Introduction: In order for cell biologists to examine the properties of cellular compartments protocols had to be formed in order to extract specific organelles from the cell. What scientists found was that if you centrifuge broken down cells at a certain centrifugal force the organelles can be separated based on their density. This method of separating organelles is known as fractionation, and is used often by scientists to study the components of cells. In this experiment, there will be an isolation of mitochondria, by fractionation of cauliflower cells, in order to prove if fractionating cells is actually successful in extracting mitochondria from the rest of the organelles in the cell. First, the broken cauliflower cells must be centrifuged.…show more content…
A BCA assay is used to measure the total protein content of the samples tested. Using a working reagent and 1x PBS a number of test tubes will be created with different concentrations of protein in order to make the BCA assay that will provide a standard curve. The BCA graph will plot the known concentrations of each of the samples against the absorbance, which is found by using the spectrometer, and a treadline will be produced. This standard curve will then be used as a model to find the concentration of proteins for each of the fractions collected during the centrifugation. In this lab, specifically the protein concentration of the nuclear supernatant (NS), mitochondrial supernatant (MS), and mitochondrial pellet (MP) will be…show more content…
About 15ml of the post-mitochondrial supernatant (MS) was removed and added to a conical tube on ice. The remaining pellet (MP) was then re-suspended using 10ml of the cold mito isolation buffer and was put into a conical tube on ice for the next class. In a previous lab samples of different protein concentration were created and labeled vials A-I in order to create a standard BCA assay that gave the equation to calculate protein concentration based off absorbance. The those samples were used to make a BCA standard curve for this lab, but instead of A-I, only the absorbances of B,C, E-I were used to create the standard curve. The absorbances were also taken for the NS, MS, and MP fractions, and the 10x dilutions of each of those fractions (twice per sample). The diluted samples were tested because the NS, MS, and MP ended up have concentrations that were off the standard curve, making their concentrations less accurate. Diluting the fractions allowed for their concentrations to appear in the range of the treadline, so those concentrations were simply multiplied by ten at the end of the lab to find their more accurate protein

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