Differential, systematic and functional proteomics. Treatment of samples. 2D-electrophoresis: IEF and SDS-PAGE. Gel analysis: ICAT, DIGE, MELANIE. 2D-HPLC. Mass spectrometry: principles, MALDI/TOF, nano-HPLC/ESI, MS/MS, imaging. Fingerprint analysis and the data banks. Other techniques: production of recombinant proteins, structural characterization of proteins
Textbooks:
Biochimica e biologia molecolare. Wilson e Walker, Raffaello Cortina editore.
Metodologie Biochimiche. Bonaccorsi, Contestabile, Di salvo. Casa editrice Ambrosiana.
Introduction to proteomics. D.C. Liebler. Humana press
Learning Objectives
Knolewdge acquired:
Preparation of samples, extraction, purification. Biochemistry of proteins. Chromatography and electrophoresis: principles and advanced techniques. Recombinant proteins and structural studies of proteins.
Competence acquired
Lessons have the aim to show the student the right methodology for the study of the proteoma from different cellular type. The analysis of the obtained results is also discussed.
Skills acquired (at the end of the course):
Students will be able to set up methodologies for the sample preparation and to perform a 2D-electrophoresis experiment.
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Prerequisites
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): 40 hours
Contact hours for: Laboratory (hours):
9 hours
Contact hours for: Laboratory-field/practice (hours):
Seminars (hours): 6 hours
Stages: 0
Intermediate examinations: 0
Further information
Office hours:
Wednesday: 11-13 a.m. and 16-18 p.m. at Dept. of Biochemical Sciences, Viale Morgagni 50
Students will be received in other days after e-mail appointment.
Type of Assessment
Exam modality:
Oral
Course program
Protein structure analyzed by bioinformatic methods
Primary structure: amino acids, structure and functional role in the proteins. Secondary structure: helix, sheet, loop, motif and domain. Classification of different folds, the protein data banks. Signal peptides, trans-membrane domains, post-translational modifications. Prediction of secondary structure. Tertiary structure: the PDB data bank.
Basic training: principles of elecrophoresis and chromatography. The western blot. The absorbance and fluorescence spectroscopy. Methods in the analysis of protein and enzymes.
Proteomics
Systematic, differential and functional proteomic: definition. Application of proteomic in the clinical, environmental, pharmacological and food analysis studies.
Methods in proteomic: preparation of samples (kind of cells and tissues, protein extraction, precipitation, reduction). The 2D-electrophoresis (isoelectric-focusing, SDS_PAGE, analysis of the results, the Melanie program). The ICAT and FIGE tecniques. The 2D-chromatography as separation technique. The Mass spectrometry of peptides and proteins: principles, instrumentation, methods (the MALDI-TOF and the HPLC-ESI-Trap instruments, the instrument of the last generation). Recovery of the proteins from the gel, the Peptide Mass Fingerprinting, identification of a protein by the mascot program. The imaging and profiling techniques and their applications.
Functional proteomic: the study of the protein-protein interaction. Mapping of the post-translational modifications; the pull-down experiments, the immuno-precipitation technique, the two-hybrid system, the use of Plasmon resonance.