User:Matt Hartings/Notebook/Photosynthesis/2012/06/30

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Objective

Purify crude sample from AA001MRH synthesis from May 16 (might have waited too long on this) and extract WT hemoglobin from yesterday's expression and continuation of Tamra's Hb cobalt-protoporphyrinIX reconstitution.

Procedure

Purification in overall synthesis of L-azidohomoalanine (full protocol found here

  1. Prep column
    1. Place glass wool in methanol to soak and place in the bottom of the 8cm diameter column
    2. Top the glass wool with ~1cm sand
    3. Prep the column material
      1. 200mL of silica (Note: next time use more ... perhaps 300mL)
      2. Prepare mobile phase 500mL of 99:1 Dichloromethane:Methanol (Overall I used around 1-1.5L for the column)
      3. Add enough of the mobile phase to the silica to make a pourable slurry
    4. Pour the silica slurry into the column
    5. Let settle for a few minutes
    6. Pressurize the column with air to pack the column and run out some of the mobile phase.
  2. Run the mobile phase through the column until there is just a little liquid remaining on top of the column.
  3. Dissolve the product (oil) from Reaction AA001MRH in the mobile phase.
  4. Add the reaction product to the column with a pasteur pipette.
  5. Run the product into the column until there is no more liquid left on the top of the column. (Don't let it dry)
  6. Add ~ 2cm of sand to the top of the column.
  7. Add more mobile phase to the top of the column
  8. Run the mobile phase through the column and collect fractions. (I collected 22 fractions)
  9. Run TLC on the fractions
  10. A compound appeared in fractions 6-15 (with the same RF) (this volume amounted to roughly 250mL)
    1. Note: the Nature protocol paper from above mentions that the product should have a RF of around 11%. My fractions appeared to have 2 different chemicals in solution with RFs of ~20% and 50%.
  11. Rotovap fractions
  12. Rotovapping resulted in an oil (as suggested by the Nature Protocol paper) of a similar volume to the oil that I began the purification with (judging by eye).
  13. The oil (in a 500mL round bottom flask) was stoppered and placed in the fridge.

Extraction of Hemoglobin

  1. Thaw cells
  2. Sonicate on power level 12
    1. 30 seconds on
    2. 30 seconds on ice
    3. repeat twice
  3. Add cells to centrifuge tubes and balance to ~0.01g
  4. Centrifuge for 2 hours at 18000rpm and 4C
  5. Prepare dialysis buffer (25mM Tris, 50mM NaCl, pH 8.3)
    1. 12.114 g Tris
    2. 11.688 g NaCL
    3. 4L water
    4. Adjust pH with 3M HCl
  6. Place Hb in dialysis bags and add to dialysis buffer
  7. Leave in cold room overnight

Continuation of Hb Co-protoporphyrinIX reconstitution

  1. Tamra had already made the Co-protoporphyrinIX solution to be added.
  2. I added this solution dropwise to the hemoglobin solution in the cold room and covered with aluminum foil


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