Protein Purification by Column Chromatography

Following the removal of the gross contaminants from the crude extract, the remaining protein components must be resolved. The most popular and ubiquitous technique for this separation is column chromatography. Similar to batch purification, column chromatography utilizes chemical and biological properties of the protein for its purification, but produces greater resolution.The background section of this chapter will cover some of the routine column chromatographic techniques used in protein purification. This discussion will elaborate on the material presented previously in Chapter 4 concerning chromatographic theory and methods.In the last experiment, α-galactosidase was captured from the crude extract and assessed for its purity. The increase in specific activity of the enzyme over the original supernatant demonstrates an initial purification. The enzyme must now be further purified to apparent homogeneity prior to its analysis. The experiment for this chapter will be to purify α-galactosidase by ion exchange column chromatography using a step gradient of sodium chloride to elute the enzyme. The α-galactosidase will be assessed for purity by determining its specific activity.