User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/08/01

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Effect of Temperature on wildtype Asc Hb Spectra Reconstituted with Nickel

  1. The 6-cell unit temperature was adjusted to 25°C.
  2. 850μL of buffer was mixed with 150μL of the concentrated wildtype Asc Hb with Ni(protoporphyrin IX) in a quartz cuvette.
  3. The cuvette was placed in the spectrophotometer and given time to cool.
  4. A spectrum from 800-200nm was taken of the protein in buffer.
  5. The temperature was increased to 30°C and a spectrum was taken again.
  6. This was repeated at 40°C, 50°C, 60°C, and 70°C.
  7. The 6-cell unit temperature was adjusted to 20°C.
  8. A spectrum from 800-200nm was taken of the protein in buffer.
  9. Spectra were also taken from 200-800nm at 10°C and 3°C.
    • There was a problem with "fogging" on the sides of the cuvette with the lower temperatures, so the cuvette was removed and wiped with a Kimwipe before the readings at lower temperatures were taken.
  10. A spectrum from 800-200nm was taken of just the buffer (25mM Tris, 50mM NaCl, pH 8) at 25°C to be used as a baseline.

Effect of Temperature on wildtype Asc Hb Spectra Reconstituted with Manganese

  1. The 6-cell unit temperature was adjusted to 25°C.
  2. 850μL of buffer was mixed with 150μL of the concentrated wildtype Asc Hb with mn-porphyrin in a quartz cuvette.
  3. The cuvette was placed in the spectrophotometer and given time to cool.
  4. A spectrum from 800-200nm was taken of the protein in buffer.
  5. The temperature was increased to 30°C and a spectrum was taken again.
  6. This was repeated at 40°C, 50°C, 60°C, and 70°C.
  7. The 6-cell unit temperature was adjusted to 20°C.
  8. A spectrum from 800-200nm was taken of the protein in buffer.
  9. Spectra were also taken from 200-800nm at 10°C and 5°C.
    • There was a problem with "fogging" on the sides of the cuvette with the lower temperatures, so the cuvette was removed and wiped with a Kimwipe before the readings at lower temperatures were taken.
  10. A spectrum from 800-200nm was taken of just the buffer (25mM Tris, 50mM NaCl, pH 8) at 25°C to be used as a baseline.


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