User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/22

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Q-Sepharose column

The remaining protein solution was placed in the fridge with 50mM Tris buffer at pH 8.3

  1. The Q-sepharose column was attached to the FPLC
  2. The protein solution was loaded in 50mL at a time
    1. the elute was collected during injection
  3. After each 50mL, a gradient of 0-160mM NaCl was run in Tris pH 8.3
    1. Fractions were collected during the gradient
      1. For the first 50mL, fractions 4-10 were collected
      2. For the second 50mL, fractions 15-21 were collected
  4. Fractions 1-3, 11-14 and 22-23 were collected in a separate tube for testing
  5. Between each injection, the column was cleaning with 1M NaCL and reequilibrated with 20mM Tris at pH8.3
  6. The column was cleaned and equilibrated with 20% Ethanol solution and stored

SP-Sepharose column

  1. The SP-Sepharose column was installed
  2. The filter to the FPLC was also changed
  3. The two sets of fractions were concentrated at 3680rpm at 4C in centrifugal devices with pH7.2 phosphate buffer 4 times in order to change the pH
  4. The SP-Sepharose column was equilibrated with pH7.2 phosphate buffer
  5. The two sets were loaded on the column and the run-through was collected
  6. The column was cleaned using 1M NaCl in tris pH8.3
    1. the elute was collected for testing purposes



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