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

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Purifying Hemoglobin

Today we will try a new method to purify our protein This will take place through FPLC using both a Q-Sepharose and a SP-Sepharose columns

  1. Spin down pH 10 protein from last week with 20mM Tris buffer at pH7.4
    1. This step is repeated three times at 3000rpm for an hour each time
  2. Make buffers for FPLC
    1. 1L 20mM Tris pH8.3
    2. 1L 20mM Tris with 1M NaCL pH8.3
    3. 1L 10mM Sodium Phosphate buffer pH 7.2
    4. 1L 10mM Sodium Phosphate buffer pH 8.0
  3. Equilibrate Q-Sepharose column with 20mM Tris buffer pH8.3
  4. Inject 10mL sample of protein into column
    1. Collect elute during injection
  5. Once loaded, run a gradient from 0 to 160mM NaCl in Tris pH 8.3 over 10 minutes
    1. This did not seem to elute anything, concentration of NaCl was risen to 300mM over 10 more minutes
    2. Fractions are collected during gradient
      1. These fractions were placed in a centrifuge filter with 50mL 10mM pH 7.2 phosphate buffer for 1 one hour at 4000rpm and 4C
      2. The initial and final elutes from the column (brown) were stored in the fridge along with the rest of the protein that wasn't used
  6. The SP-Sepharose column was attached and equilibrated with 10mM pH7.2 sodium phosphate buffer