Ammonium Sulfate Lab Report

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

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