User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/14

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Reconstitute Asc Hb with Nickel

The following procedure took place in a cold room (4°C):

  1. The apoprotein from the dialysis tubing was poured into a beaker and placed on a stir plate with a stir bar.
  2. 4mL of 1M Potassium Phosphate buffer (pH 7.1) was added dropwise (very slowly) to the protein (while gently stirring) over a 20 minute period.
  3. Ni(protoporphyrin IX)solution was added dropwise (pretty slowly) to the apoprotein (while gently stirring).
    • The Ni(protoporphyrin IX)solution was prepared by dissolving 2mg of Ni(protoporphyrin IX) in 10 drops of 0.1N NaOH, and then adding 4.5mL of dH2O. 2mL of 0.1M Potassium Phosphate buffer (pH 7.1) was added to this solution. The solution was mixed on a stir plate.
  4. The beaker with the apoprotein/Ni(protoporphyrin IX) mixture was wrapped in aluminum foil and left to gently stir in the cold room overnight.

Reconstitute Asc Hb with Manganese

  • After the Asc Hb with Manganese sat undisturbed for about 24 hours, the protein was poured into centrifuge tubes and spun at 18000rpm for two hours at 4°C.
  • The supernatent was then poured into dialysis tubing and placed in 25mM Tris, 50mM NaCl, pH 8 buffer for the rest of the weekend (with stirring).
    • This buffer was prepared fresh today by combining 6.06g Tris with 5.84g NaCl in dH2O, adding HCl to pH 8, and then diluting to a final volume of 2L with dH2O.