User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/03

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Procedure

Concentration of the protein in CHES pH10 buffer was continued for another round

  1. 10mL of the concentrated solution was loaded in the FPLC
  2. The column was stabilized with 25mL of pH10 CHES buffer
  3. The protein was injected into the column
    1. At this point, the elute is being collected in a falcon tube
    2. Once the 10mL were loaded, 5mL of CHES buffer were run through to see if anything would come off
  4. A three step NaCL gradient was run through the column to separate compounds
    1. 0-200mM NaCl over 5 minutes
    2. Stay at 200mM NaCl for 3 minutes
    3. 200mM to 500mM over 5 minutes
  5. The peaks were observed in the third step but there seemed to be more than one compound contributing to the peak
  6. This was repeated by changing the third step to be 200mM-500mM over 20 minutes
    1. This did not produce the results we were hoping for
    2. There were no observable peaks
    3. The fractions were stored until a new method can be designed