Purification of Proteins by Chromatofocusing

INTRODUCTION This protocol describes purification of proteins by chromatofocusing, a technique that separates proteins on the basis of differences in their isoelectric point (pI). The equipment required for chromatofocusing includes a chromatography column, pump, sample valve, UV monitor, pH monitor, and fraction collector. Commercially available liquid chromatography systems or high performance liquid chromatography (HPLC) systems can be used for chromatofocusing experiments. Precise pH control is essential for correct interpretation of chromatofocusing results. With a pH monitor connected to a flowthrough pH electrode, it is easy and convenient to monitor the elution pH values for chromatofocusing. Continuous monitoring of the pH profile throughout an experiment often helps explain the UV peak profiles more fully.