AIMS AND LEARNING OUTCOMES

The course provides students with a description of the laboratory procedures and instruments most commonly used in a modern biochemistry laboratory, covering the following topics: techniques for the production of cell cultures; techniques for the study and manipulation of DNA; techniques for the purification and study of proteins, with special reference to chromatographic and electrophoretic techniques.

In detail, the aim of the Course is to provide students with the basics of cellular biochemistry, macromolecules and enzymes, with an analytical approach to the main biochemistry and molecular biology techniques in use in both basic and applied research laboratories. State-of-the-art biochemical methodologies are presented and critically discussed with the 'training objective of forintaining knowledge of techniques useful in the separation, identification, and analysis of biomolecules for research, and applied applications. Course content is constantly updated.

The learning outcomes that the instructor's review of students will assess will be adequate knowledge and understanding of the biochemical techniques exhibited.

TEACHING METHODS

The course consists of classroom lectures.  Frontal lectures and examinations may be conducted remotely in telematic mode.

Lectures are developed with the support of computer materials and, as far as possible, organized by giving space to the participatory mode.

 Supporting and in-depth materials (ppt presentations, articles, links to sites of interest, bibliography, etc.) will be made available on aulaweb.

SYLLABUS/CONTENT

Applied Biochemistry- (5 CFU) Program.

Buffer solutions and pH: choice of buffers for biochemical use.

Cell cultures.

Biological samples. Homogenization: choice of methods and media.

Centrifugal techniques: principles, angular velocity, rpm, RCF, sedimentation coefficient. Centrifuges and rotors. Differential centrifugation: separation and analysis of subcellular fractions. Centrifugation on density gradient.

Spectroscopic techniques: UV/VIS spectrophotometry. Lambert-Beer's law. Determination of protein concentration by Bradford's method. NADH/NADPH absorption spectra. Enzyme activity assay of major enzymes of clinical interest: LDH, CK, succinic DH. Spectrofluorimetry (general principles and applications) and Luminometry. Assay of ATP content by the Luciferin/Luciferase method.  Mention of flow cytofluorimetry and "cell sorting".

Chromatographic techniques: general principles and applications: analytical and preparative methods. Resolution and theoretical plates. Types of low-pressure column chromatography: ion exchange, exclusion (gel filtration). Affinity chromatography. And immunoaffinity.  Mention of HPLC.

Electrophoretic techniques: general principles. Apparatus for different media (cellulose acetate, Agarose, polyacrylamide). Electrophoresis of proteins on polyacrylamide gels under native conditions and in the presence of sodium dodecyl sulfate (SDS-PAGE). PM determination of proteins. Western blotting in the study of proteins.

Gel and nitrocellulose protein staining methods.

Protein purification techniques. Purification protocols. Specific activity. Fractional precipitation of proteins: by ammonium sulfate (salting-out); by heat. Edman sequencing. Dialysis and ultrafiltration: principles.

Electrochemical techniques. Oximetry; Oxygen electrode; examples of Oxygen consumption studies.

Radioisotopic methods. Principles, instrumentation for measuring radioactivity. Mention of types of radioactive decay. Autoradiography. Mention of applications of radioisotopes in biochemistry: study of metabolic pathways.RIA.

Immunochemical techniques. Structure of antibodies. Polyclonal (antisera) and monoclonal antibodies. Immunoprecipitation. RIA; enzyme immunoassays: ELISA.

Proteomic techniques and mass spectrometry. Proteomics and Metabolomics. Isoelectrofocusing (IEF) and isoelectric point determination of proteins. Two-dimensional electrophoresis (2-DE). Colorimetric detection by Coomassie Brillant Blue and Blue silver. Mention of protein sequencing by mass spectrometry and the more moderate spectrometers quli the quadrupole.

Molecular biology techniques. Recombinant DNA technology: general principles. DNA sequencing. Identification of DNA and RNA sequences: hybridization (Southern blotting and Northern blotting). nod to CRISPR_Cas9 Technique.

RECOMMENDED READING/BIBLIOGRAPHY

Recommended reference textbook

M.C. Bonaccorsi di Patti, R. Contestabile, M.L. Di Salvo, "Biochemical Methodologies." Zanichelli Ed., 2019

Other reference texts:

M. Stoppini, V. Bellotti. Applied biochemistry EdiSES 2012

C. De Marco, C. Cini, "Principles of Biochemical Methodology," ed. Piccin, Padova, 2009