A multi-week purification protocol, suitable for students in a variety of biochemistry-related fields, is described. Working in small teams, students purify the enzyme catalase from different sources of mammalian blood via a series of three column chromatographic protocols over the course of a 5-6 week period. Elution profiles for protein and enzyme activity are assessed using rapid and cost-effective semiquantitative microscale plate assays. Students utilize these assays to critically evaluate the results of each chromatographic procedure and use this analysis to optimize subsequent steps in their purification plan. Following completion of the isolation protocol students perform quantitative assays on reserved samples from each step in the purification. These data are used to prepare a purification table to aid in assessing the efficacy of the experimental protocol. Samples of catalase at each stage of the purification are also submitted to electrophoretic analysis in denaturing and native-gel formats. This allows for additional assessment of protein purification and relative catalytic activity using commercially available low-cost digital scanning analysis software. The purification suite typically produces samples of catalase that exhibit a purification of up to 700 fold with a recovery of enzyme activity that exceeds 55 percent. Coomassie blue stained SDS-polyacrylamide gels reveal a single major protein “band” indicating a high level of purity in the final catalase product. Students gain experience in carrying out typical biochemical procedures such as chromatographic separations, enzyme and protein assays, and electrophoretic analyses while sharing and evaluating large data sets with colleagues in a laboratory team environment.

Catalase purification from mammalian blood: Development of an intensive multi-week protein purification project lab for undergraduate laboratory courses in biochemistry and related disciplines

ACS Spring 2022

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