HIC scouting

Scouting methods - Scouting for a suitable condition and HIC column for purification of A.v. plastocyanin

Scouting for conditions and the right columns is a very time consuming process. In order to facilitate the process it is done using “miniature” columns in this case 1 ml prepacked columns.

In principle it can be done on the table top using the columns and disposable syringes. However, in this course we do it using the ÄKTA start system for us to have documentation in forms of chromatograms and for us to work with linear gradients.

To our disposable we have the HiTrap HIC Selection Kit consisting of five 1 ml columns as follows

  • HiTrap Phenyl HP

  • HiTrap Phenyl FF (low sub)

  • HiTrap Phenyl FF (high sub)

  • HiTrap Butyl FF

  • HiTrap Octyl FF

We are basically trying to do the same type of experiment as described in the HIC Handbook page 29 and onwards.

We only have one set of the columns, so you have to share among the teams.

As this is a scouting exercise there is no protocol. Use some aliquots of the plastocyanin you purified yesterday. Maintain the pH at 6.5 and add a suitable amount of ammonium sulfate. Test one or two columns and see whether you can get the plastocyanin to bind and see whether you can get it to elute on a linear gradient.

The drawback of this experiment is that we are using already purified protein, but it illustrates the principle.