User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/02

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Procedure

  1. The protein solution from yesterday was placed in the centrifuge for 30 minutes with ph10 CHES buffer at 4C at 3000rpm
  2. Once concentrated, more ph 10 CHES buffer was added to the solution and placed in the centrifuge again similarly to step 1
  3. The concentrated protein solution was injected in the Q column
    1. It appeared that the protein was going straight through the column but a 50% of the salt solution over 5 minutes eluted contents of the column
    2. 7 5mL fractions were collected during the gradient
  4. A gel was run with a ladder, the original protein solution, the initial elute and fractions 4-7 from the column

CHESpH10withProteinDML07 02 14.jpg Lane 1: Ladder with BSA and Myoglobin (With nick in corner and broken gel) Lane 3: Original Protein solution in pH10 CHES buffer Lane 5: Solution eluted from column while protein was being loaded Lane 7: Fraction 4 Lane 8: Fraction 5 Lane 9: Fraction 6 Lane 10: Fraction 7 Lane 12: Ladder with BSA and Myoglobin

Preparations for tomorrow

Purified protein in pH5 acetate buffer from June 23rd

  1. Place solution in concentrating centrifugal tubes with pH10 CHES buffer
    1. Step repeated twice
    2. The tubes were placed in the fridge overnight