Preparation of samples, extraction, purification. Spectrophotometry, fluorescence. Enzymatic methods: Km, Vmax, Ki; assay of enzymes and metabolites. Immunological techniques: Chromatography: resolution, theoretical plates; absorption, partition, ionic exchange, affinity.Recombinant proteins. Electrophoresis (SDS-PAGE, IEF, 2D-EF, western blot). Mass spectrometry: principles, main sources and analyzers. Principles of proteomics (systemic, differential and functional) and metabolomic
Principles and techniques of Practical Biochemistry di K. Wilson and J. Walker. Cambridge Eds.
Learning Objectives
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.
Frontal lessons.
Practical experiences in laboratory: enzymatic and immunological assays, Western blot techniques,
HPLC and FPLC Chromatography. £Expression of recombinant proteins.
Further information
The course is formed by a first part (approx 4 ECTS) dealing with the major techinques used in a biochemistry laboratory and by a second part (approx 2 ECTS9 dealing some principles of bioclinical biochemistry (whose techniques are explained in the first part of the course)
Type of Assessment
Final test consiosts of an oral examination. The student must demonstrate that he has acquired either the knowledge of the basics of the main techniques and the ability to apply these techniques to solve problems.
Course program
Centrifugation techniques.
The coefficient of sedimentation. The factor g and the rpm. Kind of rotors. Differential, zone and isopicnic centrifugation.
Spectroscopic tecniques.
Spectrophotometry: ultraviolet and visible absorbance (principles and applications, methods of protein assay). Circular Dichroism spectroscopy. Basic principles of X ray crystallography and Nuclear Magnetic Resonance for studies on protein structure.
Spectrofluorescence and luminescence (principles, instrumentation, applications – , fluorescent probes, the GFP, other fluorescent synthetic molecules, the fluorescence microscopy).
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.
PURIFICATION AND CHARACTERIZATION OF PROTEINS
principles of protein chemistry: lysis of the
tissue and cells, fractional precipitation of proteins, salting out techniques, filtration dialysis. Choice of a specific assay method.
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 and GC techniques: instruments and applications.
Adsorption, partition, gel filtration and affinty chromatography: stationary phases, elution systems, applications. The TLC. Advanced techniques in partition chromatography (RP-HPLC and its application in different areas of research) and affinity chromatography (immunochromatography and related techniques).
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)
Notions of Clinical Biochemistry: Markers of inflammation, Clinical Enzymology (enzymes, isoenzymes, marker of infarction, marker of liver damage). Non-protein nitrogenous compounds, Plasma proteins, proteinuria.