Question

In a practical course I used an Biorad Profina system to purify a protein with a histidine tag. The device uses a column for IMAC and one for gel filtration. Why are these two devices combined in one? Why is there a need for gel filtration? I missed my chance to ask this in the practical training, thats why I ask here!

Thanks for your help!

Answer 1

The Biorad Profina system combines an IMAC column and a gel filtration column for purifying proteins with a histidine tag. The IMAC column captures the histidine-tagged proteins while the gel filtration column separates proteins based on their size.

Step-by-step explanation:

The Biorad Profina system combines an IMAC (Immobilized Metal Affinity Chromatography) column and a gel filtration column for purifying proteins with a histidine tag. The IMAC column is used to capture the histidine-tagged proteins based on their affinity to the metal ions attached to the column. However, there may still be impurities or small molecules present in the captured proteins. This is where the gel filtration column comes in.

The gel filtration column separates proteins based on their size. When the captured proteins from the IMAC column are passed through the gel filtration column, larger proteins will be excluded from the column and elute first, while smaller proteins and molecules will be retained longer in the column.

By combining these two columns, the Biorad Profina system allows for a more thorough purification of the histidine-tagged proteins by removing impurities and unbound small molecules.