User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/01

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Procedure

The Solution from yesterday was run through the anion exchange column

  1. The column was cleared from ethanol by running 1L distilled water through it
  2. The column was stabilized with 1L pH10 CHES buffer
  3. The filtered solution was placed on the column
  4. 100mL of pH 10 CHES buffer were run after the protein
    1. Nothing came off and there was a dark band about 1in thick at the top of the column
  5. 200mL of 200mM NaCL in pH10 CHES buffer were run through the column
    1. Red band started to move and was collected in a erlenmeyer flask
      1. This solution was concentrated by running in the centrifuge for 30 minutes at 3000rpm and 4C
      2. The solution was placed in a centrifugal device with a membrane that lets salts and liquid through but not protein
  6. The concentrated solution was desalted by adding more pH 10 CHES buffer and placing it in the centrifuge again
    1. This step was repeated twice