APPLIED BIOCHEMISTRY AND PROTEOMICS
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- Versione italiana
- Academic year
- 2021/2022
- Teacher
- ALESSIO BRANCHINI
- Credits
- 6
- Curriculum
- Biologia molecolare e cellulare
- Didactic period
- Primo Semestre
- SSD
- BIO/10
Training objectives
- The study of the theoretical bases, instrumental implementations and major experimental approaches in biomedical sciences, as well as preparation and analysis of samples for biochemical studies.
Knowledge and understanding.
The student:
- knows the physical and chemical principles underlying the main techniques and instruments used in biochemical investigations;
- knows the parameters that can act to modify a biochemical investigation;
- is able to understand and optimize research protocols and their parameters;
- is able to describe the structural elements of the main instruments of a biochemistry laboratory;
- knows the proper terminology used in biochemical methodologies.
Ability to employ acquired knowledge and understanding.
The student will be able to:
- use his/her knowledge to evaluate experimental results, to identify critical issues (including pros and cons) and to optimize experiments and experimental parameters;
- evaluate the impact of changing parameters involved in biochemical experiments as well as in major clinical investigations;
- identify and select the most appropriate qualitative and/or quantitative methodologies;
- properly use the terminology used to describe biochemical methodologies. Prerequisites
- There are no prerequisites.
A solid background in Biochemistry, General Chemistry and Laboratory of Chemistry, some bases of Physics, as well as Molecular Biology and Immunology are recommended to achieve a complete understanding of the proposed program. Course programme
- FIRST PART OF THE COURSE – Basic techniques (about 32 hours):
After an introduction on the topics of the Applied Biochemistry and Proteomics course, general principles, instrumentations and applications of the major experimental approaches will be provided.
- Basic techniques for the manipulation of recombinant DNA and for the production of recombinant proteins; main techniques of cell culture (transient and stable expression of recombinant proteins)
- Electrophoretic techniques (agarose gel electrophoresis, SDS-PAGE, Native-PAGE, IEF, 2D, capillary electrophoresis).
- Protein and nucleic acid purification.
- Chromatographic techniques (ion exchange, gel filtration, affinity, immunoaffinity).
- Centrifugation techniques.
- Principles, instrumentations and applications of spectrophotometric techniques (UV-visible, infrared); fluorescent probes and substrates for enzymatic kinetics.
- Structural and functional studies of proteins.
- Antibodies, features and production of monoclonal and polyclonal antibodies.
- Immunochemistry, principles and techniques (immunoprecipitation and co-immunoprecipitation, immunodiffusion, SRID, immunoelectrophoressi Rocket, Western Blotting and evolution of the method).
- ELISA principles and associated techniques (competitive ELISA, ELISPOT).
- Concepts of proteomics and examples (applied to the coagulation laboratory).
SECOND PART OF THE COURSE (about 10 hours):
Advanced techniques for fluorescent/luminescent and protein interaction studies.
- Use of reporter genes (i.e. GFP) in fluorescence microscopy.
- Flow cytometry.
- Simultaneous multiple dosages through the Bioplex system.
- Interaction studies with Amplified Luminescent Proximity Homogeneous Assay (ALPHA).
- Surface plasmon resonance (SPR).
THIRF PART OF THE COURSE (about 6 hours):
Protein engineering approaches and biotechnological applications.
- The coagulation cascade and coagulation factors.
- Plasma protein with prolonged half-life.
- Design, optimization and characterization of fusion proteins for therapeutic purposes. Didactic methods
- The main biochemical techniques will be discussed, both in theoretical and practical aspects. The course is structured in frontal lectures provided by taking advantage of power-point slides and of the classical backboard (some concepts, examples and exercises). Examples and problems will be discussed with the students. The course is designed for the understanding of the technology and scope of the proposed biochemical techniques, and for their employment for industrial and research purposes. Students are also encouraged to develop their critical thinking and communication skills through stimulation of active participation to the lessons.
Learning assessment procedures
- The exam is aimed at verifying the level of knowledge of the topics of the course program and the reasoning skills developed by the student. The evaluation at the end of the course will consist of a written exam. The assessment is expressed in thirtieths (minimum grade 18/30).
The exam will cover the theoretical and practical aspects, as well as the applications, of the main subjects and methodologies of the course. The examination will allow students to demonstrate their proficient and flexible knowledge, in terms of capacity to integrate and connect topics and concepts of the biochemical methodologies. The answers will be judged both for the content and for the appropriate scientific language.
The written exam will consist of a few (usually 3 or 4) questions, two proposing experimental patterns for comments/explanation. The students have 90 minutes for the exam. Reference texts
- Slides, schemes and figures provided.
The main topics can be found in the (italian or english) books listed below.
Books in Italian:
- Biochimica Applicata. M. Stoppini e V. Bellotti. Ed. Edises. 2012.
- Metodologie Biochimiche e Biomolecolari. M. Maccarrone. Ed. Zanichelli, 2019
- Metodologie Biochimiche. M.C. Bonaccorsi di Patti, R. Contestabile, M.L. Di Salvo. Zanichelli, 2019.
- Biochimica e Biologia Molecolare. Principi e tecniche. Ottava Edizione. Wilson & Walker. Raffaello Cortina Edizione, 2019.
Books in English:
- Principles and Techniques of Biochemistry and Molecular Biology. Eighth Edition. Wilson & Walker. Ed. Cambridge University Press. 2018.
- Molecular Biomethods Handbook. Second Edition. J.M. Walker, R. Rapley. Ed. Humana Press. 2008.
