Methods in Biochemistry

Methods in Biochemistry

Teachers: Paolo Iadarola, Gianni Francesco Guidetti

Course content

Basic knowledge of biochemistry and molecular biology. Knowledge of the general concepts about protein structure and function.

Aim of the course is to provide students with the basic knowledge of techniques typically applied in a biochemical laboratory.

a) Methods for extraction and purification of proteins from animal/vegetal tissues and cultured cells. Procedures typically used in industry/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 electrophoresis (SDS-PAGE). Isoelectrofocusing (IEF). Two-dimensional (2-DE) electrophoresis: application to proteomic studies. 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) Radioisotopes: principles, manipulation, types of emission, detection and measurement of radiation. Use of radioisotopes in biochemistry. Immunochemical techniques: production and purification of polyclonal and monoclonal antibodies and their application in biochemistry. Radioimmuno assay (RIA) and enzyme-linked immunosorbent assay (ELISA): applications in industry, clinical biochemistry and environmental monitoring.

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