APPLIED BIOCHEMISTRY
Written test with multiple choice questions and oral exam in a different date
This course aims to raise knowledge of the principles and purposes of a variety of biochemical methodologies for the purification, identification, and characterization of biological macromolecules, and for their structural and functional study. The student will be able to use the logical procedures and experimental methodology that are characteristic of scientific research, illustrate the principles and purposes of some relevant biomolecular technologies, describe and indicate their main applications. At conclusion of the training activity the student should be able to design experiments, to identify appropriate techniques for their execution, to indicate possible control experiments, to expose issues with scientific rigor and clarity, to read and critically interpret scientific publications on the subject.
Experimental method. Experimental models in biochemical research. Safety in the laboratory. Analytical and preparative separation techniques. Principles and applications of spectrofotometry, spectrofluorometry, ultracentrifugation, chromatography, electrophoresis, isoelectricfocusing, and immunochemical techniques. Specific topics include two-dimensional techniques for the protein studies, differential expression proteomics and functional proteomics, image analysis, identification of protein by mass spectrometry, PMF, LC-MS/MS
More in detail:
Experimental method. Experimental models in biochemical research. Safety in the laboratory.
Analytical and preparative separation techniques. Protein purification for structural and functional characterization.
Centrifugation techniques: Basic principles of sedimentation, preparative and analytical ultracentrifugation, density gradient ultracentrifugation.
Spectroscopic techniques: Principles and applications of Ultraviolet and visible light spectroscopy, Fluorescence spectroscopy (FRAP, FRET), Luminometry, Light scattering (Flow cytometry, FACS), Enzyme assays.
Electrophoretic techniques : General principles, Gel Electrophoresis of proteins and nuclei acids, SDS-PAGE, Isoelectricfocusing. Capillary electrophoresis.
Proteomic analysis: Two-dimensional techniques for protein studies, differential expression proteomics and functional proteomics, image analysis, identification of proteins by mass spectrometry, peptide mass fingerprint.
Chromatographic techniques : Principles of chromatography, High-performance liquid chromatography, Adsorption chromatography, Partition chromatography, Ion-exchange chromatography, Size exclusion chromatography, Affinity chromatography. Thin layer chromatography.
High performance liquid chromatography.
Chromatographic performance parameters.
Immunochemical techniques: Immunoassay formats, western blot, ELISA, Immunoprecipitation.
Maccarrone M, "Metodologie biochimiche e biomolecolari", Zanichelli Ed (I edizione, giugno 2019).
Banaccorsi MC, Contestabile R, Di Salvo ML, "Metodologie Biochimiche", Zanichelli Ed (II edizione, giugno 2019).
Wilson K., Walker J. “Principles and Techniques of Practical Biochemistry”, Cambridge University Press, fifth edition
Supplementary material (Proteomics):
Powerpoint (provided by the teacher)
Cap 12-La proteomica in biologia molecolare- in “Argomenti di Biologia Molecolare” Melino G, Ciliberto G., Società Editrice Universo
Recommended reading: Gorg A, Weiss W, Dunn MJ. “Current two-dimensional electrophoresis technology for proteomics”. Proteomics 2004.
Frontal lessons by media system presentation.
Readiness to offer individual assistance in English language to incoming students; availability of supporting material (slides and selected reviews) in English language.