View source for 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.


[[category:Protein_Preparation]]
[[category:Equipment_Usage]]