User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/29

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Preparing sample

  1. Concentrate protein solution
    1. Place in centrifugal device at 3000g, 4C for 1 hour and 15 minutes
    2. Centrifuge again with pH 8.3 20mM Tris added for a total volume of 60mL
    3. Centrifuge until the solution is reduced to ~10mL

Purification

  1. Inject solution into Q-Sepharose column
    1. Collect elute during injection
  2. Run a 0-300mM NaCl in Tris gradient over 30 minutes and collect fractions
  3. Combine desired fractions with added 20mM pH 8.3 Tris buffer
  4. Concentrate solution in centrifuge
  5. Inject solution into Q-Sepharose column
    1. Collect elute during injection
  6. Run a 0-300mM NaCl in Tris gradient over 60 minutes and collect fractions
  7. Combine desired fractions with added 10mM pH 7.2 Phosphate buffer
    1. Two sets of fractions were collected in separate tubes corresponding to two peaks
  8. Concentrate solution in centrifuge



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