Learning outcomes of the course unit
During this course , the student will be provided with
systematic notions on the main techniques used in the biochemical laboratory. Such notions concern methodologies for the identification ,
isolation, and the structural and functional study of biological macromolecules,
as well as intellectual tools for the analysis of results and their description. The acquired notions are checked during an oral examination.
Basic courses in Physics, Chemistry and Biochemistry.
Course contents summary
Basic properties of proteins related to Biochemical Methodologies: protein isoelectric point (pI), protein solubility as a function of pH and ionic strength, "unfolding" of proteins in the presence of denaturing agents (thermal and acidic denaturation, denaturation by detergents).
Subcellular fractionation by using centrifugation techniques.
Strategies used for the isolation and purification of proteins obtained from natural sources or in recombinant form.
Principles and applications of basic methodologies dedicated to proteins, for either analytical or preparative purposes: spectroscopic, electrophoretic and chromatographic techniques.
Antibodies: structure-function relationship and the immune response. Antigens and haptens. Preparation of monoclonal and polyclonal antibodies. Immunochemical methods: immunoprecipitation, ELISA techniques and Western blotting. Outline on the use of antibodies for diagnostic and therapeutic purposes.
Enzyme technology. Methods for the measurement of enzyme activity. Steady state enzyme kinetics (model of Michaelis-Menten): determination of kcat, Km and kcat/Km, and correlations with the properties of enzymes. Reversible and irreversible enzyme inhibitors, and their biotechnological relevance (drugs, pesticides ...). Examples of biotechnological use of enzymes.
Radioisotopes as biological tracers.
Methods of analysis of protein-ligand interactions: spectrophotometric and fluorimetric titrations; centrifugation and equilibrium dialysis methods. Graphical methods for the analysis of protein-ligand interactions: Scatchard-plot and Hill-plot.
Methods of analysis of protein-protein and protein-DNA interactions: techniques based on affinity chromatography, protein fragment complementation assays (yeast two-hibrid), co-immunoprecipitation, cross-linking of protein-protein and DNA-protein complexes (chromatin-immunoprecipitation), size-exclusion chromatography, DNA electrophoresis (band shift assay).
Main methodologies used in Proteomics studies.
Bonaccorsi di Patti, Contestabile, Di Salvo. Metodologie biochimiche, Casa Editrice Ambrosiana
Reed, Holmes, Weyers & Jones, Metodologie di base per le scienze
Oral lectures, with the use of multimedia means. There will also be visits to the biochemical laboratory, during which students will have the opportunity to view the use of basic techniques regarding: extraction of proteins and DNA from the cells; chromatographic separation of proteins; electrophoretic analysis of proteins and DNA; spectrophotometric analysis of protein.
Assessment methods and criteria
The acquired notions and the ability to use them in practice will be verified through an oral examination. In this test the ability to present clearly and correctly ideas and analysis will be evaluated.