Teaching code

4S02696

Credits

4

Coordinator

Not yet assigned

Language

Italian

Scientific Disciplinary Sector (SSD)

BIO/10 - BIOCHEMISTRY

Moodle Seminars 0

To show the organization of the course that includes this module, follow this link:  Course organization

The teaching is organized as follows:

Learning outcomes

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MM: teoria
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Knowledge of methodologies and methodological schemes for the study of biomolecules
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MM: laboratorio
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Knowledge and experimentation of basic biochemical methodologies, through practical exercises. The student will receive systematic training on the main techniques used in the biochemistry laboratory for the identification, isolation and structural and functional study of biological macromolecules, with particular attention to the purification and identification of proteins.
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MM: laboratorio
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Knowledge and experimentation of basic biochemical methodologies, through practical exercises. The student will receive systematic training on the main techniques used in the biochemistry laboratory for the identification, isolation and structural and functional study of biological macromolecules, with particular attention to the purification and identification of proteins.

Program

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MM: teoria
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Protein purification Ionic properties of amino acids and proteins. Isoelectric point. Sample preparation. Methods of cell disruption and production of the initial crude extracts. Protein solubilization methods. Saline swabs. Fractionation and precipitation techniques. Filtration, dialysis, sample concentration. Spectroscopic techniques. Properties of electromagnetic radiation. Light-matter interaction. States and processes involved in the phenomena of absorption, emission and decay. Absorption spectroscopy in ultraviolet and visible. Qualitative and quantitative aspects of light absorption. Colorimetric and spectroscopic methods applied to the determination of protein concentration. Spectrophotometers. Fluorescence and emission spectroscopy. Intrinsic and extrinsic fluorophores. Green fluorescent protein Spectrofluorimeters. Fluorescence resonance energy transfer (FRET). Circular dichroism. Chromatographic techniques. Principles of chromatography. The chromatogram. Parameters that determine the chromatographic performance. Van Deemter equation. Column chromatography: ion exchange, molecular exclusion, affinity, hydrophobic interaction and their applications. Electrophoretic techniques. General principles and electrophoretic mobility. Support materials. Nucleic acid electrophoresis. Protein electrophoresis. SDS PAGE. Electrophoresis in native conditions. Protein coloring on gel. Protein blotting (western blotting). Isoelectrofocusing. Basics of: Capillary electrophoresis. Two-dimensional electrophoresis gel.
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MM: laboratorio
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1. Determination of the concentration of an unknown protein by absorption at 280 nm and by Bradford colorimetric method. 2. Determination of the kinetic parameters, Michaelis-Menten constant, turnover number, and inhibition constant of the acid phosphatase enzyme using the Lineweaver-Burk graphical linearization method. 3. Determination of the absorption spectrum of the pyridine coenzyme NADH (reduced form) and determination of the molar extinction coefficient of NADPH. 4. Determination of the molecular weight of an unknown protein by molecular exclusion chromatography. 5. Protein separation by electrophoresis under denaturing conditions (SDS-PAGE) followed by visualization of the bands by Coomassie Blue staining 6. Transfer of proteins onto the nitrocellulose membrane by electroblotting followed by immunodetection of the proteins for the identification of one or more proteins by exploiting the specificity of binding with an antibody (WESTERN BLOT).
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MM: laboratorio
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1. Determination of the concentration of an unknown protein by absorption at 280 nm and by Bradford colorimetric method. 2. Determination of the kinetic parameters, Michaelis-Menten constant, turnover number, and inhibition constant of the acid phosphatase enzyme using the Lineweaver-Burk graphical linearization method. 3. Determination of the absorption spectrum of the pyridine coenzyme NADH (reduced form) and determination of the molar extinction coefficient of NADPH. 4. Determination of the molecular weight of an unknown protein by molecular exclusion chromatography. 5. Protein separation by electrophoresis under denaturing conditions (SDS-PAGE) followed by visualization of the bands by Coomassie Blue staining 6. Transfer of proteins onto the nitrocellulose membrane by electroblotting followed by immunodetection of the proteins for the identification of one or more proteins by exploiting the specificity of binding with an antibody (WESTERN BLOT).

Bibliography

Examination Methods

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The final (written) exam will focus on all the topics of the program. The student will have to demonstrate that they understand and be able to use the fundamental concepts of each topic.
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MM: laboratorio
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The written exam of Analytical Biochemistry will contain questions related to laboratory experiences
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MM: laboratorio
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The written exam of Analytical Biochemistry will contain questions related to laboratory experiences