Protein analysis using real‐time PCR instrumentation: Incorporation in an integrated, inquiry‐based project

Abstract

Instrumentation for real-time PCR is used primarily for amplification and quantitation of nucleic acids. The capability to measure fluorescence while controlling temperature in multiple samples can also be applied to the analysis of proteins. Conformational stability and changes in stability due to ligand binding are easily assessed. Protein structure studies possible with a real-time PCR instrument address core topics in biochemistry and have valuable highthroughput applications in the fields of drug discovery and protein engineering. Protein analysis using real-time PCR instrumentation has been incorporated in an undergraduate laboratory project based on previously described projects. Students express, purify, and characterize a protein.Based on literature research and analysis using bioinformatics tools, they select a specific mutation to investigate. They then attempt to express, purify, and characterize their mutated protein. Thermal denaturation using a real-time PCR instrument is the primary tool used to compare the wild-type and mutated proteins. Alternative means for incorporation of protein analysis by real-time PCR instrumentation into laboratory experiences and additional modes of analysis are also described. V C 2013 by The International

Inhibitory effects of flavonoids isolated from Sophora flavescens on indoleamine 2,3-dioxygenase 1 activity

“…The thermal stability assay has been widely used to confirm the interactions between compounds and their target proteins. The interaction of compound is expected to enhance the thermal stability of the protein 29 , 30 . The binding of representative flavonoids ( 4 – 6 ) to IDO1 was determined by the temperature stability analysis using SYPRO-Orange.…”

“…The binding of representative flavonoids ( 4 – 6 ) to IDO1 was determined by the temperature stability analysis using SYPRO-Orange. The SYPRO-Orange binds to the hydrophobic residues in the unfolded regions of proteins, and is denatured by temperature 30 . Then we exposed the flavonoids ( 4 – 6 ) to IDO1 with SYPRO-Orange in order to confirm whether they change unfolding temperature of IDO1 enzyme without L -Trp.…”

Doing that thing that scientists do: A discovery‐driven module on protein purification and characterization for the undergraduate biochemistry laboratory classroom

“…This approach extends upon previous work showing that inquiry-based instruction benefit students' understanding and retention in STEM fields [7][8][9][10]. Others have developed similar pedagogies with success [11][12][13]. We have expanded upon this success with this module; students are given a research topic, basic research techniques and the independence to implement them to answer questions that they develop in the course of their lab work.…”

Guided inquiry activity linking thermodynamic parameters of protein unfolding to structure using differential scanning fluorimetry data in the biophysical chemistry classroom

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