Biochemical methodologies
(objectives)
TRAINING OBJECTIVES The Teaching Course of Biochemistry Methodologies aims to provide the students with a theoretical and practical knowledge on some of the major preparative and analytical methods that are used not only in biochemical and molecular biological research, but also in other fields including biomedical and environmental. In particular, the knowledge provided relates to methodologies for the identification, isolation and structural and functional characterization of biological macromolecules as well as intellectual tools for the analysis of results and for their description. Both techniques for analyzing individual proteins and genes, as well as whole proteomes and genomes will be treated.
EXPECTED LEARNING OUTCOMES Knowledge and understanding: At the end of the course the students 1) will know the basics of the main techniques used in biochemical investigations (centrifugation, spectroscopy, electrophoresis, chromatography, spectrometry, protein and nucleic acids sequencing) and the parameters to change in order to improve the results of a specific biochemical investigation; 2) will be able to describe the structural elements of the main instruments of a biochemical laboratory (centrifuge, spectrophotometer, spectrometer); 3) will know the appropriate terminology used in biochemical methods.
Applying knowledge and understanding: At the end of the course the students will be able to: 1) orient themselves to choosing the most appropriate biochemical procedure for achieving the experimental goals defined during the research design; 2) evaluate the possible impact of variations in the key parameters of a biochemical experiment; 3) practically carry out the experiments performed during the practical part of the course.
Making judgements: Students should be able to understand and discuss critically the experimental results obtained in a research laboratory and use them as the basis for planning subsequent experiments.
Communication skills: Students should have the ability to convey the acquired knowledge in a clear and comprehensible manner, even to people who are not competent, and must demonstrate the ability to present information also with graphs and formulas.
Learning skills: Successful condition in learning is the ability to read and understand a scientific paper on biochemical topic.
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Code
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17531 |
Language
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ITA |
Type of certificate
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Profit certificate
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Credits
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6
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Scientific Disciplinary Sector Code
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BIO/10
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Contact Hours
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40
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Laboratory Hours
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8
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Type of Activity
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Related or supplementary learning activities
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Teacher
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IMPERLINI ESTHER
(syllabus)
Theoretical part (40 hours): - The principles of biochemical research. - Buffer systems. Ionization of amino acids and proteins as a function of pH. - Error theory. Accuracy and precision. - Centrifugation: basic principles of sedimentation; centrifuges and their use; rotors; separation methods in the preparative centrifugation; differential centrifugation; density gradient centrifugation; analytical ultracentrifugation. - Strategies for isolation and purification of proteins; cellular extraction methods; dialysis and ultrafiltration; fractional precipitation with salts, with organic solvents, to the isoelectric point, to heat. - Spectrophotometry: laws and applications; Lambert-Beer law; absorption spectrum of nuclei acids and proteins; spectrophotometric determination of protein concentration: Biuret, Lowry and Bradford methods; enzyme dosing. - Chromatographic methods: adsorption and partition chromatography, affinity, molecular exclusion, ion exchange, hydrophobic interaction, reverse phase liquid chromatography, HPLC. - Electrophoresis: theory and laws; classification of electrophoretic methods; equipment for electrophoresis gel; electrophoresis of agarose, acrylamide and SDS-PAGE; techniques of detection and staining of electrophoretic gels; classical two-dimensional electrophoresis (2-DE) and Differential in Gel Electrophoresis (DIGE technology). - Immunochemical techniques: western blot; immunoprecipitation and immunoaffinity as strategies for the analysis of protein complexes and protein interactions. - Mass spectrometry: principles and application in modern proteomic analysis.
Practical Part (8 hours): - Determination of the protein concentration of an unknown sample using the Bradford method. Spectrophotometer display of absorption spectrum of a protein with/without prosthetic groups; discussion of results.
- Preparation of a polyacrylamide gel in the presence of SDS (SDS-PAGE) and electrophoretic run with pre-stained molecular weight markers, in order to follow the protein mobility during the electrophoretic process. Illustration of devices for the preparation of agarose gel and/or western blotting.
(reference books)
- Wilson K., Walker J. Principles and techniques of practical biochemistry. 5th ed., 2000, Cambridge University Press.
Handouts are provided by the teacher for practical laboratory activities.
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Dates of beginning and end of teaching activities
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From 26/02/2023 to 08/06/2023 |
Delivery mode
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Traditional
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Attendance
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not mandatory
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Evaluation methods
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Oral exam
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