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:
- 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.