Preparation of samples, extraction, purification. Spectrophotometry, fluorescence and chemioluminescence. Enzymatic methods: Km, Vmax, Ki; assay of enzymes and metabolites. Immunological techniques: Chromatography: resolution, theoretical plates; absorption, partition, ionic exchange, affinity. Electrophoresis (SDS-PAGE, IEF, 2D-EF, western blot). Mass spectrometry: principles, main sources and analyzers. Principles of proteomics (systemic, differential and functional).
Textbooks:
Biochimica e biologia molecolare. Principi e tecniche. di K. Wilson and J. Walker. Raffaello cortina editore
Metodologie di base per le scienze biomolecolari- R. Reed, D. Holmes, j. Weyers, A. Jones. Zanichelli editore
Learning Objectives
Knolewdge acquired:
Preparation of samples, extraction, purification. Spectrophotometry: absorbance and fluorescence. Chromatography and electrophoresis: principles and advanced techniques. Production and characterization of recombinant proteins. Laboratory methods: Assay of enzymes and metabolites in clinical and environmental practise. Immunological analysis of hormones, antigens, antibodies. The set up and management of a biochemical laboratory. Proteomics techniques: 2D electrophoresis and 2D-chromatography, mass spectrometry. Differential and functional proteomics. Clinical and environmental application of Proteomics.
Competence acquired
Lessons have the aim to show the student the right methodology for the analytical and preparative determination of proteins, enzymes, metabolites, hormones. The analysis of the obtained results is also discussed. Theoretical and practical aspects of a proteomic study.
Skills acquired (at the end of the course):
Students will be able to set up experiments of protein purification, enzyme assay, electrophoresis of DNA and proteins, western blot. 2D electrophoresis techniques and sample preparation.
Prerequisites
Courses to be used as requirements (required and/or recommended)
.
Courses required: Biochemistry. Experimental techniques in biology
Courses recommended: Biochemistry. Experimental techniques in biology. Cellular biochemistry. Molecular biology
Courses to be used as requirements (required and/or recommended)
.
Courses required: Biochemistry. Experimental techniques in biology
Courses recommended: Biochemistry. Experimental techniques in biology. Cellular biochemistry. Molecular biology
Teaching Methods
Total hours of the course (including the time spent in attending lectures, seminars, private study, examinations, etc...): 150
Hours reserved to private study and other indivual formative activities:
Contact hours for: Lectures (hours): 48
Contact hours for: Laboratory (hours): 12
Contact hours for: Laboratory-field/practice (hours):
Seminars (hours): 0
Stages: 0
Intermediate examinations: 0
Further information
Frequency of lectures, practice and lab:
the frequency is strongly suggested.
Teaching tools
The instrumentation of the Department of Biochemical Sciences is using to perform demonstrative lessons and practice.
Office hours:
Wednesday: 11-13 a.m. and 16-18 p.m. at Dept. of Biochemical Sciences, Viale Morgagni 50
Students can be received in other days after e-mail appointment.
Type of Assessment
oral examination about the course contents and critical lecture of a scientific paper.
Course program
Principles
Units measurements, buffer solutions, quantitative analysis. Calibration methods, internal standards.
Structural characterization of amino acids and of primary, secondary and tertiary protein structures.
Centrifugation techniques.
The coefficient of sedimentation. The factor g and the rpm. Kind of rotors. Differential, zone and isopicnic centrifugation. The analytical centrifugation.
Spectroscopic tecniques.
Spectrophotometry: ultraviolet and visible absorbance (principles and applications, methods pf protein assay). Spectrofluorescence and luminescence (principles, instrumentation, applications – immunological assay, fluorescent probes, the GFP, other fluorescent synthetic molecules, the fluorescence microscopy).
Recombinant proteins
Cloning, expression and purification of proteins in bacteria, yeasts, eukaryote cells. Structural and functional characterization of recombinant proteins.
Chromatography, electrophoresis and mass spectrometry in proteomics
Definition and application of proteomics. The systematic, differential and functional proteomic.
Chromatography
Principles: partition coefficient, theoretical plates. Instrumentation. The HPLC technique.
Adsorption, partition, gel filtration and affinty chromatography: stationary phases, elution systems, applications. The TLC. Advanced techniques in partition and affinity chromatography.
Electrophoresis
Principles. Agarose and PolyAcrylamide. Preparation of samples. SDS-PAGE, , western-blot and immune-blot. Isoelectric focusing.
2D-electrophoresis: principle, instrumentation, application (the proteomics)..
Mass spectrometry
Sensibility, specificity. The mass spectrum. The M+ and the parent ions. Instrumentation. The ion source: electronic impact; the soft-ionization (the Fast Atom Bombardment, the Matrix-Laser-Desorption-Ionization, the Electro-Spray-Ionization). Analyzers: magnetic and electric sectors, the quadrupole, esapole, ion-trap, the FT-MS analyzer, the Time-of-Flight analyzer.
The Gas-Cromatography-mass-Spectrometry instrument. The MALDI-TOF, nano-HPLC-ESI instruments. Application in the proteomics (systematic and differential proteomic). The MS/MS spectrometer for the determination of peptide sequence.
Other proteomics techniques
The SELDI, ICAT and DIGE techniques for the differential proteomic.
Main techniques in functional proteomics (post-translational modifications, protein-protein interactions, pull-down assay and two hybrid system)
Metods in the biochemistry laboratory
The enzymatic methods for the quantitative assay of enzymes and metabolites. The principles of the methods: the end point and the kinetic method (principles of enzymatic kinetic). The use of synthetic substrates and of the coupled reactions.
Some examples: transaminases, creatin-kinase, deidrogenases, glucose, cholesterol, triglycerides.
The immuno-enzymatic methods: RIA, IRMA, ELISA, CL and fluorescence. Principles of the competitive, sandwich and indirect method. Marcature of antigens and antibodies . Sensitivity and specificity of the technique. Examples: TSH, T3/T4, PTH, cytokines, CMV.
The problem of certification and quality control.