User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/16

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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:
  1. 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. 2.5mL of the dialyzed Reconstituted Asc Hb was then run over the column.
  3. The protein was eluted off with 3.5mL of 25mM Tris, pH 8.3.
  4. 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:
  1. 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. 2.5mL of the dialyzed Reconstituted Asc Hb was then run over the column.
  3. The protein was eluted off with 3.5mL of 25mM Tris, pH 8.3.
  4. 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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