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B016589 - APPLIED BIOCHEMISTRY
Academic Year 2019-20
Course year
Third year - Second Semester
Belonging Department
Chemistry "Ugo Schiff"
Course Type
Single education field course
Scientific Area
BIO/10 - BIOCHEMISTRY
Credits
9
Teaching Hours
72
Teaching Term
02/03/2020 ⇒ 13/06/2020
Attendance required
No
Type of Evaluation
Final Grade
Course Content
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Course program
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Lectureship
Course Content
Cell cultures. Homogenization and centrifugation. Spectrophotometry and fluorimetry: principles and applications. Chromatographic methods. Electrophoresis. Radioactive isotopes. Monoclonal- and polyclonal antibodies. Enzymes and metabolites assay. Immobilized enzymes. Recombinant DNA technology
Course program
Introduction to course. Approaches to the experimentation and the development of strategies for purification. Main substrate used in the experiments. Cell cultures: Primary and stablished cell lines. Contact inhibition and cell counting. Culture media, incubators and laminar flow hoods. Homogenization techniques in solid or liquid media. Blenders, Potter and Ultra TURRAX. Centrifugation techniques. Centrifugal field and speed centrifugation. Sedimentation coefficient. Differential centrifugation, zonal velocity and isopicnic centrifugation. Rotors used.
Spectrophotometry: Lambert and Beer's law and its applications. Colorimetric methods for the determination of protein. Photometers and spectrophotometers.
Fluorescence Spectroscopy: Principles of physical phenomenon; instrumentation, and applications. Chromatographic techniques: Partition coefficient and theoretical plates. Column chromatography on paper and thin layer. Different types of chromatography: ion exchange, excluding and affinity. Electrophoresis of proteins and nucleic acids: the principle of the technique; SDS-PAGE; Isolectrofocusing, capillary electrophoresis. Purification and determination of the molecular weight of protein.
Radioisotopes: radioactive decay law. Emitters , γ. Determination of radioactivityà by the ionization of a gas, scintillation and the spark autoradiography
Polyclonal antibodies: immunization and production of antisera. Properties of monoclonal antibodies. Radioimmunoassay (RIA) and enzyme immunoassay (EIA).
Enzymes: qualitative and quantitative analysis. Measure of initial velocity and specific activity. Mixing dosing. Determination of kinetic parameters. Dosage of metabolites in the diagnosis. Immobilized enzymes.
Nucleic acids: sequencing, and chemical synthesis. Recombinant DNA technology: Restriction enzymes, cloning and expression vectors. In vitro amplification of DNA (PCR). Enzymatic synthesis of cDNA by mRNA. Production of recombinant proteins: somatostatin, insulin, growth hormone, vaccines. Gene therapy techniques
Spectrophotometry: Lambert and Beer's law and its applications. Colorimetric methods for the determination of protein. Photometers and spectrophotometers.
Fluorescence Spectroscopy: Principles of physical phenomenon; instrumentation, and applications. Chromatographic techniques: Partition coefficient and theoretical plates. Column chromatography on paper and thin layer. Different types of chromatography: ion exchange, excluding and affinity. Electrophoresis of proteins and nucleic acids: the principle of the technique; SDS-PAGE; Isolectrofocusing, capillary electrophoresis. Purification and determination of the molecular weight of protein.
Radioisotopes: radioactive decay law. Emitters , γ. Determination of radioactivityà by the ionization of a gas, scintillation and the spark autoradiography
Polyclonal antibodies: immunization and production of antisera. Properties of monoclonal antibodies. Radioimmunoassay (RIA) and enzyme immunoassay (EIA).
Enzymes: qualitative and quantitative analysis. Measure of initial velocity and specific activity. Mixing dosing. Determination of kinetic parameters. Dosage of metabolites in the diagnosis. Immobilized enzymes.
Nucleic acids: sequencing, and chemical synthesis. Recombinant DNA technology: Restriction enzymes, cloning and expression vectors. In vitro amplification of DNA (PCR). Enzymatic synthesis of cDNA by mRNA. Production of recombinant proteins: somatostatin, insulin, growth hormone, vaccines. Gene therapy techniques