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Methods in Biochemistry

Corsi di laurea:
Molecular Biology and Genetic
Docenti:
Iadarola Paolo
Anno accademico:
2009/2010
Codice corso:
500802
Crediti formativi:
9
Ambito:
BIO/10
Decreto Ministeriale:
270/04

Programma

Aim of the course is to provide students with the basic knowledge of the techniques applied in a typical biochemical laboratory in which extraction, purification and characterization of proteins, used as model-molecules, is performed.

a) Methods for extraction and purification of proteins from animal/vegetal tissues and cultured cells. Description of procedures typically used in industry and research laboratories for primary purification of proteins. Theoretical principles and practical protocols of the most common chromatographic approaches : adsorption; partition; ion-exchange (IEC); gel-filtration (SEC); affinity; hydrophobic-interaction(HIC); perfusion; gas-chromatography (GC) and supercritical-fluid chromatography (SFC).Chromatofocusing; high performance liquid chromatography (HPLC) ; fast protein liquid chromatography (FPLC) and principles of green chromatography.
b) Electrophoretic techniques: analytical and preparative monodimensional (1-DE) electrophoresis of proteins in their native and denaturated state. Sodium-dodecyl-sulfate gel elctrophoresis (SDS-PAGE) for the determination of protein molecular weight. Two-dimensional (2-DE) electrophoresis: application to proteomic studies. Isoelectrofocusing (IEF). Capillary Zone electrophoresis (CZE) and Micellar Electrokinetic Chromatography (MEKC).
c) Spectroscopic techniques for the qualitative/quantitative characterization of proteins.
Adsorption spectroscopy: ultraviolet (U.V).; Visible; Infrared (IR); Nuclear Magnetic Resonance (NMR); Electron Spin Resonance (ESR). Emission spectroscopy: fluorescence and phosphorescence. Mass Spectrometry (MS).
d) Study of the primary sequence of a protein.
Amino acid analysis with pre-column and post-column derivatization. Automated sequence degradation. Chemical and enzymatic cleavage of proteins. Labeling of the active site with radioactive probes. Methods of detection of radioactivity.
e) Radioimmunological (R.I.A.) and immunochemical (ELISA) techniques for the determination of antigens/antibodies in clinical and environmental use.

Bibliografia

Principles and Techniques of Practical Biochemistry by K. Wilson and J. Walker. 5th Edition. Cambridge University Press.


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