User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/21

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Purifying hemoglobin

  1. Collect frozen protein solution from last week and thaw
  2. Once thawed, the solution's pH was changed using concentration device for centrifuge
    1. 15mL of protein solution was added to 45mL of pH8.3 50mM Tris buffer
    2. The solutions were spun at 3000rpm for 30 minutes
  3. Repeat the last step
    1. The solutions were spun until the final total volume was less than 40mL
  4. Spin down concentrated protein solution to remove impurities
  5. Equilibrate Q-Sepharose column with 50mM Tris buffer pH8.3
  6. Inject 10mL sample of protein into column
    1. Collect elute during injection
  7. Once loaded, run a gradient from 0 to 160mM NaCl in Tris pH 8.3 over 10 minutes collected in 5mL fractions
    1. A peak was collected in this range
    2. tube labelled fractions 11,12,13
    3. concentration is raised to 300mM
    4. Another set of fractions is collected and placed in tube labelled fractions 14-23
      1. These fractions were placed in a centrifuge filter with 50mL 10mM pH 7.2 phosphate buffer for 1 one hour at 3500rpm and 4C
      2. Repeated twice keeping the two sets of fractions separate
      3. 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
  8. The SP-Sepharose column was attached and equilibrated with 10mM pH7.2 sodium phosphate buffer
  9. 10mL of each set of fractions were loaded separately
  10. The run-through was collected in tubes labeled with the corresponding fractions

All solutions stored in fridge overnight



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