A protein purification suite, suitable for upper-division biochemistry or biotechnology undergraduate students, is described. Students work in small teams over the course of 5 weeks to isolate the enzyme catalase from mammalian blood using a sequence of column chromatographic procedures. Protein and enzyme activity elution profiles are determined by graphical analysis of assay data collected using simple, rapid, inexpensive and visually appealing semiquantitative microplate assays. Students are able to quickly perform a real-time critical evaluation of their data to optimize the preparation of starting material for subsequent steps in the purification scheme. After performing quantitative assays on final purification “pools”, students use these data to construct a purification table for use in evaluating the experimental protocol. Electrophoretic separations are carried out in both denaturing and native-gel formats. Native gels are visually examined after completion of a colorful “negative staining” procedure to detect relative catalase activity in each purification pool.  Denaturing gels, stained with Coomassie blue to detect protein, are analyzed visually and by use of commercial digital analysis software to further evaluate the purification of catalase. The protocol may also be modified in a number of ways to serve as a starting point for research-style small-group projects later in the term.<br/>

Purification and characterization of catalase from mammalian tissue: Development of a multi-week protein purification and characterization project lab for upper division biochemistry and biotechnology laboratory courses

254th National Meeting (2017)

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