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.
Purification in overall synthesis of L-azidohomoalanine (full protocol found here
- Prep column
- Place glass wool in methanol to soak and place in the bottom of the 8cm diameter column
- Top the glass wool with ~1cm sand
- Prep the column material
- 200mL of silica (Note: next time use more ... perhaps 300mL)
- Prepare mobile phase 500mL of 99:1 Dichloromethane:Methanol (Overall I used around 1-1.5L for the column)
- Add enough of the mobile phase to the silica to make a pourable slurry
- Pour the silica slurry into the column
- Let settle for a few minutes
- Pressurize the column with air to pack the column and run out some of the mobile phase.
- Run the mobile phase through the column until there is just a little liquid remaining on top of the column.
- Dissolve the product (oil) from Reaction AA001MRH in the mobile phase.
- Add the reaction product to the column with a pasteur pipette.
- Run the product into the column until there is no more liquid left on the top of the column. (Don't let it dry)
- Add ~ 2cm of sand to the top of the column.
- Add more mobile phase to the top of the column
- Run the mobile phase through the column and collect fractions. (I collected 22 fractions)
- Run TLC on the fractions
- A compound appeared in fractions 6-15 (with the same RF) (this volume amounted to roughly 250mL)
- 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%.
- Rotovap fractions
- 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).
- The oil (in a 500mL round bottom flask) was stoppered and placed in the fridge.
Extraction of Hemoglobin
- Thaw cells
- Sonicate on power level 12
- 30 seconds on
- 30 seconds on ice
- repeat twice
- Add cells to centrifuge tubes and balance to ~0.01g
- Centrifuge for 2 hours at 18000rpm and 4C
- Prepare dialysis buffer (25mM Tris, 50mM NaCl, pH 8.3)
- 12.114 g Tris
- 11.688 g NaCL
- 4L water
- Adjust pH with 3M HCl
- Place Hb in dialysis bags and add to dialysis buffer
- Leave in cold room overnight
Continuation of Hb Co-protoporphyrinIX reconstitution
- Tamra had already made the Co-protoporphyrinIX solution to be added.
- I added this solution dropwise to the hemoglobin solution in the cold room and covered with aluminum foil