Teacher
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BUONOCORE Francesco
(syllabus)
Buffer systems. Ionization of amino acids and proteins as a function of pH. The principles of biochemical research. Error theory. Accuracy and precision.
Spectrophotometry: laws and applications. Lambert-Beer law. Absorption spectrum of nuclei acids and proteins. Spectrophotometric determination of protein concentration.
Centrifugation. Basic principles of sedimentation. Centrifuges and their use. Rotors. Separation methods in the preparative centrifugation. Differential centrifugation. Density gradient centrifugation. Analytical ultracentrifugation.
Strategies for isolation and purification of proteins. Cellular extraction methods. Dialysis and ultrafiltration. Fractional precipitation with salts, with organic solvents, to the isoelectric point, to heat.
Chromatographic methods: adsorption and partition chromatography, ion exchange, molecular exclusion, affinity, hydrophobic interaction, HPLC, gas chromatography. Efficiency of chromatographic procedures and selection of a convenient method.
Electrophoresis: theory and laws. Classification of electrophoretic methods. Free phase and zonal electrophoresis. Equipment for zonal electrophoresis gel. Gel electrophoresis under native and denaturing conditions (SDS-PAGE). Application of SDS-PAGE. Western blotting. Isoelectrofocusing: principles, equipment and methods. Two-dimensional gel electrophoresis. Capillary electrophoresis.
Studies on the structure of proteins. Determination of amino acid composition, free cysteines, amino acid sequence and disulphide bridges.
Mass spectrometry: principles.
(reference books)
WILSON, WALKER: Biochimica e biologia molecolare. Principi e tecniche.(Raffaello Cortina) De Marco, Cini: Principi di metodologia biochimica (Piccin) Bonaccorsi, Contestabile, Di Salvo: Metodologie biochimiche (Casa Editrice Ambrosiana) The lecturer will add power point presentations.
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