User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/28

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Clean and store Desalt column

  1. Run 150mL water through the column
  2. Run 150mL 20% Ethanol solution throughout the column

Make buffers for FPLC

  1. Prepare 400mL 20% Ethanol
    1. 80mL 200 poof ethanol in 320mL FPLC water
  2. Filter 1L H20 and prep for autoclave
  3. Prepare 1L 20mM Tris pH 8.3
    1. Filter and prep for autoclave
  4. Place buffer and water in autoclave at 121C for 1 hour

Prepare Protein solution

  1. Thaw tube on lab bench
  2. Place solution in centrifugal device with 30mL pH8.3 20mM Tris buffer
  3. Centrifuge for 30 minutes at 3000g
  4. Repeat above steps but this time for 1 hour
  5. Centrifuge until final volume is 10mL


Equilibrate Q-Sepharose column

  1. Run 25mL H2O through the column
  2. Run 25mL 20mM Tris pH 8.3
  3. Column is ready for protein

Purification fo Protein

  1. Inject protein into column
    1. Collect elute during injection
  2. Run a gradient 0-300mM NaCl in Tris over 10 minutes
    1. Collect fractions
  3. Combine desired fractions into single tube
  4. Clean column with 20mL 1M NaCl
  5. Re-equilibrate column with 20mM Tris pH 8.3
  6. Concentrate and desalt fractions using centrifugal device
    1. Add 20mM Tris to fractions twice to remove salt
    2. centrifuge until final volume is 10mL


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