Design of Purification and Analysis Protocols for Riboflavin Binding Protein in an Undergraduate Biochemistry Laboratory

Abstract

One of the common protocols of undergraduate biochemistry laboratories is the purification of a protein of interest. A protocol to purify riboflavin binding protein (RBP) was modified from published and unpublished sources for use in the undergraduate Biochemistry Laboratory course at Capital University (Chemistry 452). RBP is an excellent choice for purification in the undergraduate laboratory. Because it can be isolated from chicken eggs, no large and time‐consuming bacterial cultures are necessary. The yellow color of RBP makes it easy to follow throughout the purification protocol and less likely the students will lose the protein during the course of the purification. The purification protocol also includes many basic purification techniques, including both ion‐exchange and size exclusion column chromatography and salt precipitation. Once the purification protocol is completed, the protein can be analyzed for purity using gel electrophoresis and the ratio of riboflavin to protein can be analyzed using UV‐Vis spectrophotometry. Because of the fluorescence properties of both riboflavin and the RBP apoprotein, the unfolding properties of RBP can be easily examined.