Protein Expression
- Cells were placed in each of the 25mL Erlenmeyer flasks and left to grow overnight in a shaking incubator at 37C and 200rpm
- Each of these solutions was transferred to a 1L broth solution with 1mL of ampicilin (100mg/mL)
- The flasks were left to incubate for 3 hours at 37C on the shaking incubator at 130rpm
- After 4 hours, 1ml of Fe-protoporphyrin IX was added to each flask and the temperature was reduced to 30C and left to incubate for another 4 hours
- The Fe-protoporphyrin IX (Hemin) was made by dissolving 40mg of Fe-protoporphyrin IX in 1mL of DMSO
- 1mL of IPTG was also added to each flask
- 4mM of IPTG in H20 prepared immediately before adding to flasks
- After four hours, the flasks were placed in a cold room over night
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 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 signification followed by 30 seconds on ice until the pellet was loose and liquid
- The solution was then placed in the freezer overnight
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