Cell and Protein Extraction
- The solutions were centrifuged for 15 minutes at 4500 rpm at 4 degrees Celsius
- The pellet was collected and placed in a 50mL falcon tube
- The falcon tube was centrifuged at 4500 rpm for an additional 15 minute to precipitate the cells
- The black supernatant was removed (but not discarded) and placed into a 15mL falcon tube
- A small amount of 5mM Tris buffer was used to collect remaining traces of cells from the centrifuge tubes
- The pellet was then sonified
- 30 seconds sonification followed by 30 seconds on ice until the pellet was loose and liquid
- Once liquefied, the black supernatant was put back in the tubes and the solution was centrifuged at 20,000rpm four 3 hours at 4C
- The tubes were spun for an additional 30 minutes under the same conditions in order to prepare the materials for the next step
- 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
- Amount added was the equivalent of 5mL of powder
- The tubes were collected from the centrifuge and the supernatant was placed in a 50mL falcon tube
- Both the supernatant and the saturated ammonium sulfate solutions were placed on ice for 10 minutes
- A volume of saturated ammonium sulfate solution equivalent to 1/4 of that of the supernatant was added to the latter
- in this case, 6mL of the saturated solution were added to the 24mL of supernatant
- Notice solution become cloudy
- This mixture was left on ice for 30 minutes and then placed in the centrifuge at 4C, 10000g for 30 minutes
- The supernatant was placed in dialysis tubing and that was placed in 4L of neutral phosphate buffer overnight
- The pellet was resuspended in 50mM Tris and placed in the fridge
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