User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/10

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Cell and Protein Extraction

  1. The solutions were centrifuged for 15 minutes at 4500 rpm at 4 degrees Celsius
  2. The pellet was collected and placed in a 50mL falcon tube
  3. The falcon tube was centrifuged at 4500 rpm for an additional 15 minute to precipitate the cells
  4. The black supernatant was removed (but not discarded) and placed into a 15mL falcon tube
  5. A small amount of 5mM Tris buffer was used to collect remaining traces of cells from the centrifuge tubes
  6. The pellet was then sonified
    1. 30 seconds sonification followed by 30 seconds on ice until the pellet was loose and liquid
  7. Once liquefied, the black supernatant was put back in the tubes and the solution was centrifuged at 20,000rpm four 3 hours at 4C
    1. The tubes were spun for an additional 30 minutes under the same conditions in order to prepare the materials for the next step
  8. A saturated solution of Ammonium Sulfate was prepared by adding Ammonium Sulfate powder to 10mL of distilled H2O until it would no longer go into solution
    1. Amount added was the equivalent of 5mL of powder
  9. The tubes were collected from the centrifuge and the supernatant was placed in a 50mL falcon tube
    1. Both the supernatant and the saturated ammonium sulfate solutions were placed on ice for 10 minutes
  10. A volume of saturated ammonium sulfate solution equivalent to 1/4 of that of the supernatant was added to the latter
    1. in this case, 6mL of the saturated solution were added to the 24mL of supernatant
    2. Notice solution become cloudy
  11. This mixture was left on ice for 30 minutes and then placed in the centrifuge at 4C, 10000g for 30 minutes
    1. The supernatant was placed in dialysis tubing and that was placed in 4L of neutral phosphate buffer overnight
    2. The pellet was resuspended in 50mM Tris and placed in the fridge