Lecture 26 summary

Protein pI values and ion-exchange chromatography

Whether a particular protein sticks to an ion-exchange column or not depends on the following:

• Whether the ion-exchange column contains a cation exchange staionary phase (resin) or an anion exchange resin.
• The isoelectric point (pI) of the protein
• The pH of the mobile phase (usually a buffered solution with a specific salt concentration)
• The ionic strength of the mobile phase - high salt concentrations provide ions that compete with protein for binding sites on the ion-exchange resin.

Recall that the isoelectric point of a protein is the pH at which the net charge of the protein is zero. A polymer such as a protein, has very many groups that can act as proton donors and acceptors, and hence many different possible charge states. The figure below illustrates how the charge on a given protein will change as the pH of a proteins's environment changes in relation to the pI of the protein.