Is the performance of TALON resin affected by the temperature?

If the protein requires colder temperatures to prevent degradation, the purification steps can be performed at 4°C. This is a matter of preference, as the resin will work at either room temperature or 4°C. However, the resin should never be allowed to freeze because the agarose beads can break, leaking metal ions and decreasing binding capacity.Protein purification [...]

What is the binding capacity of TALON resin?

The binding capacity of TALON resin is 5–15 mg protein/ml of resin. This can vary, depending on the type and size of the his-tagged protein.Protein purification [...]

What should be done if the protein contains a tag in the middle of the sequence, i.e., not a terminal tag?

There is no reason that a 6xhis tag in the center of a protein would not work, as long as it is exposed after protein folding. In fact, some customers have successfully isolated proteins with a tag in this location. Even if the folding of the native protein masks the tag, it is possible that purification under denaturing conditions would work.Protein purification [...]

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 [...]

How can the yield of purified HAT- or 6xHis-tagged fusion protein be increased?

There are different factors that can affect and increase the yield of purified protein: -Temperature Always use cold (4–8°C) solutions during extraction, and centrifuge at cool temperatures as well. Performing extractions and purifications at 4°C may reduce protein degradation. -Use of protease inhibitors If proteolysis is suspected to be occurring during the extraction process, consider using [...]