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