Protein Expression
- Cells were placed in each of the 25mL Erlenmeyer flasks and left to grow overnight in a shaking incubator at 37C and 225rpm
- 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 removed from the incubator
- The solutions were centrifuged for 15 minutes at 4500rpm and the supernatant (clear broth) was discarded
- The pellets were combined into 50mL falcon tube
- Protease inhibitors were prepared
- 50mg of AEBSF . HCL [Pefabloc SC] (from Enzo life Sciences) were dissolved in 35mL 50mM Tris buffer solution
- The solution was used to collect the remaining cells from the centrifuge tubes
- The tubes containing the cells and true Tris solution were centrifuged for 5 minutes at 2500rpm
- The tubes were placed in the freezer
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