Fluorescence quantum yield measurements of fluorescent proteins: A laboratory experiment for a biochemistry or molecular biophysics laboratory course

• Published in: Biochemistry and molecular biology education Vol. 43; no. 1; pp. 52 - 59
• Main Authors: Wall, Kathryn P., Dillon, Rebecca, Knowles, Michelle K.
• Format: Journal Article
• Language: English
• Published: United States Wiley-Blackwell 01-01-2015; Wiley Subscription Services, Inc
• Subjects: Biochemistry; Biochemistry - education; Biophysics; Biophysics - education; College Science; Fluorescence; fluorescence spectroscopy; GFP; Green Fluorescent Proteins - chemistry; guided inquiry; Humans; Inquiry; interdisciplinary; Laboratory Experiments; laboratory instruction; Light; Measurement; Molecular Biology; Molecular Structure; physical chemistry; Proteins; Research - education; Science education; Science Experiments; Science Instruction; Scientific Concepts; upper‐division undergraduate; GFP; guided inquiry; laboratory instruction; physical chemistry; proteins; biochemistry; upper-division undergraduate; fluorescence spectroscopy; biophysics; interdisciplinary
• ISSN: 1470-8175; 1539-3429
• DOI: 10.1002/bmb.20837

Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts absorbed photons into emitted photons and it is necessary to know for assessing what fluorescent protein is the most appropriate for a particular application. In this work, we have designed an upper‐level, biochemistry laboratory experiment where students measure the fluorescence quantum yields of fluorescent proteins relative to a standard organic dye. Four fluorescent protein variants, enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), mCitrine, and mCherry, were used, however the methods described are useful for the characterization of any fluorescent protein or could be expanded to fluorescent quantum yield measurements of organic dye molecules. The laboratory is designed as a guided inquiry project and takes two, 4 hr laboratory periods. During the first day students design the experiment by selecting the excitation wavelength, choosing the standard, and determining the concentration needed for the quantum yield experiment that takes place in the second laboratory period. Overall, this laboratory provides students with a guided inquiry learning experience and introduces concepts of fluorescence biophysics into a biochemistry laboratory curriculum. © 2014 by The International Union of Biochemistry and Molecular Biology, 43(1):52–59, 2015.