User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/25

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Procedure

  1. Prepare a 300uM solution of sodium dithionite in 100mL of Tris buffer at pH 8.3
  2. Degas solution for an hour
    1. Nitrogen was bubbled through the solution
  3. A disposable column was obtained
    1. GE PD-10 pre-packed column
  4. The column was cleaned by running 25mL of dionized water through it
  5. The column was equilibrated by running 25mL of the degassed solution through it
  6. Once equilibrated, we started running the protein in the following steps
    1. 2.5mL of protein are added to the column (do not collect elute)
    2. 3.5mL of degassed Tris with DTT are added to the column (collect elute)
    3. 25mL of degassed Tris with DTT are added to the column to re-equilibrate (do not collect elute)
    4. Repeat the steps until all the protein has been run through the column
  7. After the first collection, a solution of Ruthenium was added to the elute and the following collations (of protein) were added to the same tube
    1. The ruthenium solution was made up to contain 10X more Ruthenium than protein
  8. Once all the protein were collected from the column, the tube was covered in foil and placed on the shaker for 4 hours
  9. During the wait period, the desalting column on the FPLC was cleaned and equilibrated with Tris buffer pH 8.3
  10. After 4 hours of reactions, the protein solution was run through the desalting column
  11. The protein and ruthenium excess were separated using the column and stored in separate tubes
  12. The tubes were placed in the freezer until further purification next week

To do next week

  1. Concentrate protein from desalting column
  2. Clean and restabilize desalt column
  3. Run through Q-column w/ gradient 0-30% NaCl 10 minutes and collect fractions
  4. concentrate elute from q-column
  5. Run through Q-column w/ gradient 0-30% NaCl 30 minutes and collect fractions
  6. concentrate elute from q-column
  7. Run through Q-column w/ gradient 0-30% NaCl 1 hour and collect fractions
  8. concentrate elute from q-column
  9. run gel