Running a Cobalt Affinity Column

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Running a Cobalt Affinity Column (Purifying a His-tagged Protein)

  1. Obtain the cobalt resin from the refrigerator and a clean column with a valve and top.
  2. Swirl the cobalt resin to create a slurry mixture.
  3. Pour a small amount of slurry (5 mL) into the gravity column. Make sure the valve is closed.
  4. Drain the storage buffer from the cobalt resin.
  5. Wash the resin with five column volumes of nanopure water (5 x 5 mL = 25 mL) to remove the all of the storage buffer.
  6. Equilibrate the resin with five column volumes of wash buffer.
  7. Add the protein mixture to the column and collect the flow through.
  8. Apply the flow through to the column again.
  9. After two passes through the resin, the flow through can be placed on ice.
  10. Wash the column with wash buffer and collect 10mL aliquots.
  11. Check the A280 of each aliquot.
  12. Once the wash has reached an A280 of about zero, only the his-tagged protein remains on the column.
  13. Elute the protein with elution buffer. Collect in 10 mL aliquots.
  14. Monitor the A280, and keep all fractions well above zero. These contain protein.
  15. Once all the protein has eluted, strip the column with 1M imidazole.
  16. Wash with 5 column volumes of nanopure water.
  17. Add 20% ethanol to the resin for storage.
  18. Place the column in the refrigerator for future use.
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