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

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Reconstituting and Purifying Asc Hb with Ni(protoporphyrin IX)

  • The protein was poured out of the dialysis tubing back into centrifuge tubes and then spun at 18000rpm at 4°C for an hour. The supernatent was decanted carefully using a Pasteur pipette so the pellet was not disturbed.
  • 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.