What should be done if a his-tagged protein is secreted from the cell into the cell culture medium?
What should be done if a his-tagged protein is secreted from the cell into the cell culture medium?
Centrifuge the medium at 4°C, 10,000g for 30 min and check the supernatant for the presence of chelating substances that could prevent protein binding to the resin, as follows: apply a small quantity of the supernatant (e.g., 10 ml), adjusted to a pH of >7, to 1 ml of resin packed in a column.
If the resin does not lose Co2+, i.e., it remains pink, it is safe to continue the purification with the rest of the sample using one of the purification protocols from the TALON Metal Affinity Resins User Manual.
If the cobalt ion is removed by chelating substances in the supernatant, then performing a buffer exchange via gel filtration, ultrafiltration, or diafiltration is recommended (the preferred technique depends upon the sample volume). HisTALON Equilibration Buffer, a PBS-based buffer (available in the HisTALON Buffer Set, Cat. # 635651), can be exchanged for the medium if it is compatible with the protein of interest.
Once the protein is in a buffer compatible with TALON resin, it should be purified using the protocol in the user manual associated with your TALON product. Recombinant protein expression in eukaryotic cells can vary widely depending on the expressed protein. If the his-tagged protein has a good expression level, then a helpful rule of thumb is to start with a 10:1 ratio of medium to resin (e.g., for 100 ml of medium you may start with 10 ml of resin). This should bind all of the secreted, his-tagged protein.