Unit BIOCHEMICAL METHODS
- Course
- Biology
- Study-unit Code
- GP004082
- Curriculum
- Bionutrizionistico
- Teacher
- Carla Emiliani
- Teachers
-
- Carla Emiliani
- Sandra Buratta (Codocenza)
- Hours
- 35 ore - Carla Emiliani
- 12 ore (Codocenza) - Sandra Buratta
- CFU
- 6
- Course Regulation
- Coorte 2019
- Offered
- 2019/20
- Learning activities
- Affine/integrativa
- Area
- Attività formative affini o integrative
- Academic discipline
- BIO/10
- Type of study-unit
- Opzionale (Optional)
- Type of learning activities
- Attività formativa monodisciplinare
- Language of instruction
- Italian
- Contents
- Working in a Biochemistry laboratory. Methods for the production of recombinant proteins. Methods for protein purification: separation, purification and analysis techniques. Spectroscopic methods for the study of proteins. Mass spectrometry. Enzymes and analysis techniques of enzymatic enzymatic activity.
- Reference texts
- Keith Wilson and John Walker: Biochimica e biologia molecolare:Principi e tecniche¿ EDs. 2019 Raffaello Cortina Editore. ¿
M.Maccarrone: Metodologie Biochimiche e Biomolecolari, Zanichelli Editore
Bonaccorsi, Contestabile, Di Salvo -Metodologie biochimiche-Casa Editrice Ambrosiana. Distrib. Zanichell - Educational objectives
- Knowledge: at the end of the course the student must know the biochemical methodologies focused mainly on the strategies and techniques of expression, purification and characterization of proteins
Capacity: at the end of the course the student must be able to understand which tools are needed to deal with the study of proteins and more generally of biological macromolecules - Prerequisites
- Basic knowledge in the field of organic chemistry and biochemistry
- Teaching methods
- Frontal lectures on the topics covered by the program. The lessons will be conducted with the help of slides and videos. Some lessons will be held in the laboratory with the help of tutors.
- Other information
- Optional but strongly advised
- Learning verification modality
- It consists of a written test that will serve to ascertain the level of knowledge and understanding of the topics covered during the course and will have a maximum duration of two hours, during which the student will have to answer multiple choice and / or open questions.
Students who have not passed the written test will have the opportunity to analyze the critical issues together with the teacher.
For information on support services for students with disabilities and / or DSA visit the page http://www.unipg.it/disabilita-e-dsa. - Extended program
- Introduction to the course; design, carry out and evaluate a biochemistry experiment; use of Internet and databases.
Working in a Biochemistry laboratory: basic instrumentation; the most common operations (stock solutions and dilutions; pH control, etc.).
Methods for the production of recombinant proteins: cloning strategies of a known coding sequence; expression systems; production systems (bacteria, yeasts, mammalian cells, insect cells); fusion proteins; use of tags; example of production of a recombinant protein.
Separation techniques: preparation of a protein extract, methods of tissue homogenization, cell rupture, subcellular fractionation; fractionation of protein mixtures, centrifugation, fractionated precipitation with salts.
Chromatography: general concepts, molecular exclusion chromatography, ion-exchange chromatography, affinity chromatography, partition chromatography, adsorption chromatography, high-performance liquid chromatography (HPLC)
Electrophoresis: general principles, protein electrophoresis in native and denaturing conditions (SDS-PAGE), isoelectric focusing, two-dimensional electrophoresis, gel staining (comassie brilliant blue, silver staining, fluorescent dyes, ethidium bromide), nucleic acid electrophoresis.
Quantitative methods: determination of protein concentration by colorimeter assays; radioisotope methods; immunochemical techniques (Western blotting, ELISA, RIA).
Spectroscopic methods for the study of proteins: UV-Visible absorption spectroscopy, the Lambert-Beer law; the chromophoric groups; calibration plots for quantitative analysis; methods for protein quantification (Bradford, biureto, BCA, Lowry); outline of fluorescence spectroscopy.
Mass spectrometry and its applications.
Enzymes, measure of enzymatic activity (Kat., UI, SA), preparation and execution of an enzymatic assay; determination of enzyme concentration, substrate concentration, Km and enzyme inhibition, temperature and pH effects. Biosensors as examples of the use of enzymatic kinetics to dose enzymes or substrates.