Reconstitute Asc Hb with Nickel
- After the Asc Hb with Nickel sat undisturbed overnight, the protein was poured into centrifuge tubes and spun at 18000rpm for two hours at 4°C.
- The protein was then purified over a PD-10 Desalting column:
- A PD-10 Desalting column was prepared with one column volume of dH2O was run over the column, and two column volumes of 25mM Tris, pH 8.3 run over the column.
- 2.5mL of the dialyzed Reconstituted Asc Hb was then run over the column.
- The protein was eluted off with 3.5mL of 25mM Tris, pH 8.3.
- The loading and elution of the protein was repeated many times.
Purifying the Asc Hb with Manganese
- The protein was first purified over a PD-10 Desalting column:
- A PD-10 Desalting column was prepared with one column volume of dH2O was run over the column, and two column volumes of 25mM Tris, pH 8.3 run over the column.
- 2.5mL of the dialyzed Reconstituted Asc Hb was then run over the column.
- The protein was eluted off with 3.5mL of 25mM Tris, pH 8.3.
- The loading and elution of the protein was repeated many times.
- Run over a HiTrap Q FF column on an FPLC in the same way done with Asc Hb with Ni(protoporphyrin IX) on 07/09/12
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