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Ammonium Sulfate Lab Report

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This experiment was not carried out in chronological order to maximize efficiency. First the volume (3.33mL) of needed liquid ammonium sulfate was calculated based on the assigned 25% saturation. 3.33mL of the liquid ammonium sulfate was added to 10mL of the protein extract (given by the instructor) in a centrifuge tube to precipitate the protein out. Water was then added to a second centrifuge tube to balance out the mass in the centrifuge. The mass of the mixture was weighed and the water was measured (using a scale) to weigh the same as the mixture. Both centrifuge tubes were place in the centrifuge for ten minutes at 12,000rpm. The supernatant was pipetted out of the centrifuge tube, leaving the protein pellet that developed during centrifuging. In the tube with the …show more content…

10mL of the protein solution (given by the instructor in the beginning of the laboratory) contained 0.3mg/mL. A blank was made using 0μL of the protein standard stock solution, 2.0mL of the Bradford reagent and 100μL of saline (5mM NaCl). Five dilutions 20μL, 40μL, 60μL, 80μL, and 100μL of the protein standard solution were prepared in cuvettes. Aaline (5mM NaCl) was added to each of the cuvettes accordingly to bring their volumes up to 100μL. Then 2.0mL of the Bradford reagent was added to each diluted solution. The blank cuvette was placed in the spectrophotometer with the arrow on the cuvette matching to the arrow inside the spectrophotometer. The spectrophotometer was zeroed on the computer at 595nm using this blank as the reference. Then one by one each cuvette with the different diluted protein solution was placed in the spectrophotometer and their absorbance was recorded. This was then plotted as a standard curve showing absorbance (y-axis) and protein in micrograms (x-axis). The x-axis of this curve was calculated based on the concentration ~0.3mg/mL of the original protein standard

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We began to test of hypothesis by preparing another set of cuvettes with our Pellet 2 cell fraction of cauliflower because it had the greatest SDH activity. This time, we added assay buffers of different pH values. The values we used to test our hypothesis were pH values of 5.5, 6.0, 7.5, and 8.5. We tested 3 different pH values and kept our 4th pH with the value of 7.5 as the control, or blank, again. We again tested for absorbance at 600nm at times 0 minutes, 7 minutes, and 14 minutes.

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Initially a 1:1:1 mixture of an unknown acid, base, and neutral compound was obtained with the goal of being separated through extraction and then further purified through recrystallization. Since the mixture was a 1:1:1 ratio, this means the amount present was .333 grams for each compound as this is important for calculating the percent recovery from extraction. The percent recovery is the percentage of the original product that is recovered, in this case the percent of the initial 0.33g that is recovered. The basic extraction using NaOH yielded a crude amount of 0.226 g of the acid. The final mass was not measured until removal of the base occurred and the compound was dried for a week.

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Results The purpose of this experiment was to identify whether or not the OAT protein within the malpighian tubules transports particles with little specificity other than the requirement that they are organic anions. This was tested through the use of CPR to examine the amount of PAH that is transported by the protein. Because an increased amount of transported PAH would result in a decreased amount of CPR within the tubules due to competition, a data point for this experiment would consist of a tubule, either incubated in a PAH or non-PAH solution, that corresponds to a shade of red within the comparison tray. This comparison tray contains 5 different concentrations of CPR, which are also different shades of red (0.00468 mM, 0.0094 mM, 0.019

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15 mL of Solution A and B were mixed together to form solution F. Eight cuvettes were labeled distinctly as 1a, 2a, 3a, 4a, 1b, 2b, 3b, 4b, where “a” cuvettes were used for the concentration experiment and “b” cuvettes were used for the temperature experiment. Cuvette 1, the blank tube was prepared and the spectrophotometer was set to 405 nm. The enzyme was added, upon being ready to start the experiment, to tube 1 which then became tube “1a.” 3 mL of solution F was added to each cuvette, both “a” and “b.” The “b” cuvettes were then placed in their specific temperatures, 1b in the fridge, 2b in room temperature, 3b in a 32 degrees Celsius water bath and 4b in a 60 degree water bath. The temperature was recorded using a thermometer that was placed in the surroundings of the tube.

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H and S were diluted 20x and P1 and P2 were diluted 2x to make up a total volume of 1ml each. 50ul of each diluted sample was pipetted into 8 wells of the microplate and 50ul of each protein standard was pipetted into 2 wells. 50ul were incubated with 50ul of alkaline copper reagent. 50ul of alkaline copper reagent was added to every well containing water, buffer, sample or standard and was incubated for 30 minutes. 100ul of Folin reagent was added to the wells and incubated for another 20 minutes.

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After 10 minutes of activity, 1.5mL of sodium carbonate was added to all of the tubes to stop the enzyme activity. Approximately 1 mL of each solution was added to a cuvette and then placed in a spectrophotometer set at 421 nm. The spectrophotometer measured the light absorbance or optical density of the solution which is depicted in figure 2. As the temperature was increased, the enzyme activity had also increased, however, after 90*C the enzyme activity dropped dramatically because it became denatured. The data supports the direct relationship of enzyme activity and temperature as well as the heat causing the enzyme to become denatured after

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We then collected 2 mL of that solution into another tube with 10mL of phosphate buffer. After developing the enzyme, we placed 1 mL of lactase into each cuvette. In another beaker, we added 1 mL of lactase in 1 mL of phosphate to create the positive control. Before using the spectrophotometer, we set it to 420 nm and blanked the machine with the cuvette that was filled with only the phosphate buffer. Right before we set the cuvettes into the spectrophotometer, we added 1 mL of ONPG to each

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The supernatant was poured into a clean, rinsed with distilled water, twenty-five milliliter graduated cylinder without pouring out the pellet. The volume of the supernatant was restored to fifteen milliliters with the homogenizing medium, shaken well and transferred to the clean tube, marked S, and kept on ice. The volume of S was exactly the same as H. Ice-cold homogenizing medium was added to the pellet to make the final volume exactly equal to the original volume centrifuged, fifteen milliliters. The tube was stopped and shaken to suspend the

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The plate is then centrifuged at 300x g for 5 minutes at 4 ºC. The supernatant is discarded and the pellet is resuspended in 20 μL antibody

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Control samples are included in procedure to confirm the experiment. Therefore, the first tube stayed the color clear and no protein is present. The second tube of albumin turned a purple color showing a protein is present. The third tube of pepsin turned a purplish/pink showing a protein is present. This happens because pepsin is an enzyme and they are composed of proteins.

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