Integrating a Research‐Based Experience in the Biochemistry I Laboratory: From Separation of Colored Compounds to Protein Purification

Abstract

As part of our CUR Transformations Project work within the Department of Chemistry and Biochemistry, we aimed to create an additional research‐based experience for the Biochemistry I laboratory course. We designed a series of chromatography experiments that focus on careful observation, experimental design, data interpretation, and presentation skills. Students first work with three different types of columns during a two‐week chromatography lab. The first week they work with two mixtures of colored compounds, each mixture contains 3 out of the 5 compounds. These two mixtures are run through gel filtration, anion exchange, and cation exchange columns. Students analyze their observations in week 1 to design a purification scheme for separation of all 5 colored compounds together in one mixture in week 2. After finishing the two‐week lab, students write a paper based on their results in the format of Nature Methods. Following this experiment, students start a 3‐week lab where they design a scheme to separate two unknown colorless proteins in a mixture. They are given the following information: isoelectric points for both proteins, one of the two proteins has a 6x histidine‐tag, and the 260 nm extinction coefficient of the tagged protein. Students then choose a cation exchange, anion exchange, or an affinity column to use for protein separation. Students analyze their chromatography procedure by using the Bradford reagent to detect protein in column fractions, denaturing gel electrophoresis to assess purification and determine molecular weights of the two proteins, and a western blot to verify identity of one of the proteins using an antibody to 6x histidine‐tags. Lastly, students present their results to their peers in week 4.Support or Funding InformationWe would like to acknowledge support for this CUR Transformations project through NSF DUE IUSE grant # 16‐25354.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.