Isolation, purification, and characterization of bovine heart and yeast cytochromes <i>c</i>: An integrated biochemistry laboratory experience

The undergraduate biochemistry laboratory curriculum is designed to provide students with experience in protein isolation and purification protocols as well as various data analysis techniques, which enhance the biochemistry lecture course and give students a broad range of tools upon which to build in graduate level laboratories or once they begin their careers. One of the most common biochemistry protein purification experiments is the isolation and characterization of cytochrome c. Students across the country purify cytochrome c, lysozyme, or some other well-known protein to learn these common purification techniques. What this series of experiments lacks is the use of sophisticated instrumentation that is rarely available to undergraduate students. To give students a broader background in biochemical spectroscopy techniques, a new circular dichroism (CD) laboratory experiment was introduced into the biochemistry laboratory curriculum. This CD experiment provides students with a means of conceptualizing the secondary structure of their purified protein, and assessments indicate that students' understanding of the technique increased significantly. Students conducted this experiment with ease and in a short time frame, so this laboratory is conducive to merging with other data analysis techniques within a single laboratory period. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):515-520, 2017.

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Circular dichroism spectroscopy: Enhancing a traditional undergraduate biochemistry laboratory experience

Lewis

Seal

Lorts

et al. 2017

“…As we began to modify our curriculum to focus on relationships between techniques, we searched for an existing project‐based curriculum that could be adapted to our needs. Purification and characterization of a protein seemed to be the obvious choice, and several successful versions do appear in the literature . However, these experiments were difficult to incorporate into our curriculum as written, because they required at minimum 4–6 hours of class time per week and were designed for a class size of 16 students or fewer.…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of enzymes from yeast: An extended undergraduate laboratory sequence for large classes

Johanson

Watt

McIntyre

et al. 2013

Providing a project-based experience in an undergraduate biochemistry laboratory class can be complex with large class sizes and limited resources. We have designed a 6-week curriculum during which students purify and characterize the enzymes invertase and phosphatase from bakers yeast. Purification is performed in two stages via ethanol precipitation and anion exchange chromatography, and students perform both direct and coupled enzyme assays. By completion of the experimental series, students are able to identify which enzymes they have purified and have obtained kinetic parameters for one. This experimental series requires minimal instructor preparation time, is cost effective, and works with multiple sections of large groups of students. Students participating in this sequence showed increases in conceptual understanding of biochemical concepts as measured through in-class assessments and anonymous surveys.

A real‐time and hands‐on research course in protein purification and characterization: Purification and crystal growth of human inosine triphosphate pyrophosphatase

Kreiling

Brader

Kolar

et al. 2011

A new lecture/laboratory course to offer advanced biochemical training for undergraduate and early graduate students has been developed in the Department of Chemistry at the University of Nebraska at Omaha. This unique course offers students an opportunity to work hands-on with modern instrumentation not normally found in a predominately undergraduate institution, and to complete an entire research project in a realistic timeframe via a time-intensive curriculum as a special summer session. The course content gives a strong background in protein structure/chemistry, purification principles, protocol development, optimization strategies, use and programming of an automated chromatography instrument, and characterization strategies with an emphasis on X-ray crystallography. The laboratory portion offers students the chance to purify a protein (human inosine triphosphate pyrophosphatase) from start to finish, program and use an Ä KTA fast protein liquid chromatography instrument, and to grow and analyze their own protein crystals using their purified protein. This innovative laboratory experience gives the participating students the opportunity to complete a miniresearch project in real time and enhances their overall understanding of important biochemical research techniques and the instrumentation involved, fostering a better understanding of the research process all within a classroom setting. Evaluations and feedback concerning this course indicated a positive learning environment, a retention of knowledge and skills, a belief that the skill set learned continues to be useful in current endeavors, and a sense of accomplishment in the completion of an actual research project within the confines of a class setting.Keywords: Active learning, curriculum design development and implementation, laboratory exercise, new course development, protein structure function and folding.One major downfall in any undergraduate laboratory course is the disconnection between a series of 3-hour miniaturized experiments and the realistic nature of true bench work. In a typical undergraduate course, the experiments are written to introduce students to working at a bench, calculations, and use of equipment, but are all designed with maximal successful outcome ratios for the students. In an actual research laboratory setting, students must deal with experiments that take extended timeframes to complete, multiple trials for optimization, and work in collaboration to optimize equipment and reagent use. Students quickly learn that research results are not as easily obtained as their former laboratory manuals made it seem. Many of our science majors at the undergraduate level are hoping to join graduate programs or work forces which require applicants with research experience, who are detailed notebook writers, and who have developed solid critical thinking skills. Graduate students may find that their research project involves the need for protein isolation and/or purification, a skill set that may be outside of their laboratory's area of...