Expression and Purification of Sperm Whale Myoglobin

Miller, Stephen T.; Indivero, Virginia; Burkhard, Caroline

doi:10.1021/ed8000842

Cited by 12 publications

(12 citation statements)

References 11 publications

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“…Second, the materials and equipment required for the detection assay, isolation steps, and final analysis are also reasonably inexpensive and currently stocked in most undergraduate laboratories. Finally, the isolation steps are designed to infuse a large number of visual cues into the isolation of a colorless enzyme from a natural product (and therefore it represents a more typical real life example compared to isolating colored proteins, such as green fluorescent protein or myoglobin (Miller et al 2010), that are over expressed in genetically modified sources). These visual cues include the following: (i) the 2-step precipitation in ethanol requires students to consider where their enzyme is located at all times (the decantate or pellet); (ii) the addition of visual markers to the sample prior to gel filtration lets them know where their enzyme is located on and off the column; (iii) contaminating blue dextran in the gel filtration enriched fraction of invertase is removed in the last isolation step by adsorption to the DEAE cellulose column, in this way they know the column is working; (iv) The fractions are first visually analyzed by SDS-PAGE, prior to performing the more technically challenging and less intuitive estimate of purity by specific activity measurements; and, finally (v) commercial sources of the pure enzyme to serve as a gold standard of comparison against their own results is readily available and inexpensive.…”

Section: Resultsmentioning

confidence: 99%

The Isolation of Invertase from Baker’s Yeast – An Introduction to Protein Purification Strategies

Timerman¹

2012

Protein Purification

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Section: Resultsmentioning

confidence: 99%

The Isolation of Invertase from Baker’s Yeast – An Introduction to Protein Purification Strategies

Timerman¹

2012

Protein Purification

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“…Additionally, plasmids are widely available, relatively inexpensive, and can be synthesized to facilitate the investigation of diverse research objectives, including several that have been previously published in this Journal . For example, students could express myoglobin for investigations of heme-containing enzymes, phospholipases and fibroblast growth factors to facilitate investigations into protein–protein interactions, or xylanase for investigations of dynamic secondary structure under changing conditions . There are also commercial kits available for the expression of recombinant fluorescent proteins and dihydrofolate reductase (Bio-Rad, Hercules, CA), both of which can be used to facilitate student-led investigations.…”

Section: Potential Variationsmentioning

confidence: 99%

Modernizing the Analytical Chemistry Laboratory: The Design and Implementation of a Modular Protein-Centered Course

et al. 2021

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Course-based undergraduate research experiences (CUREs) integrate authentic research into undergraduate chemistry laboratories, introducing students to research while simultaneously reinforcing fundamental concepts. Despite their ubiquitous nature in bioanalytical research, few CUREs have been published applying the fundamental techniques of separations, spectroscopy, quantification, and mass spectrometry. To engage students in learning these increasingly essential bioanalytical techniques, we designed and implemented a semester-long project-based course centered around the purification, quantification, and identification of heterologously expressed proteins in five succinct and adaptable modules. Instructors can use these modules to form the foundation of a CURE specific to their research interests. The extensive commercial availability of plasmids for transformation combined with the modular approach to laboratory experiments enables convenient customization to accommodate diverse research goals. Instructors can tailor the modules to meet the curricular requirements and instrumentation capabilities of their institutions and can easily extend the research goals to incorporate more specialized analytical techniques, as needed. Through the implementation of the five modules, students apply the fundaments of acid−base chemistry, statistics, quantification strategies, spectrophotometry, separations, and mass spectrometry, thus covering the material required in most undergraduate introductory analytical courses. Instructors can then use these modules as a backbone to support student-led discovery-based investigations for the remainder of the course. Students demonstrate their understanding through the completion of a comprehensive, publication-style laboratory report as well as a poster presentation at a university-wide undergraduate research symposium. Since first offering this course in 2016, student evaluations have been exceedingly positive, with over 75% of students indicating that the course both increased their scientific skills as well as their confidence in their ability to succeed in further science courses. Furthermore, 22% of students reported they were "much more" or "extremely more" likely to enroll in a Ph.D. program in science, math, or engineering following the courses, emphasizing the impact that project-based laboratories can have on undergraduate chemists' career trajectories.

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“…Size exclusion chromatography (SEC), the premier method for characterizing macromolecules in terms of molecular mass and molecular mass distribution, gives educators another approach for introducing polymer science into undergraduate chemistry programs. ,, A literature search of this Journal on SEC as a pedagogical approach for studying macromolecules has revealed a number of papers on the use of this technique in the undergraduate curriculum; − however, there are none that address SEC theory from a thermodynamic perspective.…”

Section: Introductionmentioning

confidence: 99%

Size Exclusion Chromatography: A Teaching Aid for Physical Chemistry

Barth¹

2018

J. Chem. Educ.

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All analytical techniques are governed by the principles of physical chemistry, and chromatography is certainly no exception. By examining the underlying concepts of size exclusion chromatography (SEC), students can gain greater appreciation of and insight into the science and utility of thermodynamics, as well as solution properties of macromolecules. In this paper we will show how the separation mechanism that governs SEC, an unusual form of chromatography, can be explained using the tenets of thermodynamics. This paper gives educators access to an HPLC technique usually not included in undergraduate chemistry curriculum.

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Expression and Purification of Sperm Whale Myoglobin

Cited by 12 publications

References 11 publications

The Isolation of Invertase from Baker’s Yeast – An Introduction to Protein Purification Strategies

The Isolation of Invertase from Baker’s Yeast – An Introduction to Protein Purification Strategies

Modernizing the Analytical Chemistry Laboratory: The Design and Implementation of a Modular Protein-Centered Course

Size Exclusion Chromatography: A Teaching Aid for Physical Chemistry

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