title: 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 high-throughput 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.
authors.affiliation: Department of Chemistry, Indiana University of Pennsylvania, Indiana, Pennsylvania, 15705.
authors.forename: Jonathan N
authors.initials: JN
authors.lastname: Southard
mesh_terms: D007753:Laboratories; D015202:Protein Engineering; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation*
chemical_list: D011506:Proteins
keywords: Real-time PCR; high-throughput analysis; inquiry-based laboratory; integrated laboratory project; ligand binding; protein stability; thermal denaturation