00062 - Applied Biochemistry

Learning outcomes

The aim of the Course is to give a basic knowlewdge about the major techniques and principles underlying biochemical experiments.
Techniques useful in the separation, identification, and analysis of biomolecules will be discussed.

Course contents

Course contents:

General principles of  biochemical investigations. In vivo models: animal and plant studies. In vitro studies: organ and tissue homogenates. Cell cultures. pH and buffer solutions in Biochemistry.

Cell disruption.

Centrifugation techniques: principles of sedimentation. Centrifuges and preparative rotors. Differential centrifugation: separation and analysis of subcellular fractionations. Differential flotation of lipoproteins. Density gradient centrifugation: zonal and isopycnic technique. Separation of cells, subcellular organelles, proteins, nucleic acids. Evaluation of the yield and the enrichment: subcellular markers.

Protein precipitation techniques: salt and isoelectric precipitation fractionation; fractionation with organic solvents and organic polymers. Heat precipitation.

Dialysis and ultra-filtration.

Solvent extraction.

Chromatografic techniques: chromatography theory and practice. Column chromatography: efficiency and resolution. Thin-layer chromatography (TLC).

Adsorption and partition chromatography. Hydrophobic interaction chromatography (HIC) of proteins.

Ion-exchange chromatography. Amino acid analyzers.

Molecular exclusion chromatography.

Affinity chromatography. Lectin affinity chromatography. Immunoaffinity  chromatography. Dye-ligand chromatography.  Metal chelate chromatography: purification of His-tagged proteins. Affinity techniques in the purification of recombinant proteins.

HPLC and FPLC. Sequencing of proteins.

Electrophoretic techniques: general principles. Support media: cellulose acetate electrophoresis of serum proteins. Polyacrylamide gel electrophoresis (PAGE) and PAGE-SDS. Detection and estimation of proteins in gels. Western blotting. Isoelectric focusing (IEF); titration curves of proteins. 2D polyacrilamide gel electrophoresis and applications in Proteomics. Capillary electrophoresis.

Analytical Tecniques

Spectroscopic techniques

UV and visible spectroscopy in Biochemistry. Applications. Quantitative analysis: protein assays.

Spectrofluorimetry: principles, instrumentation and applications. Intrinsic and extrinsic fluorescence. Protein and membrane structure. FRET. Fluorescence bleaching recovery (FRAP). Microspectrofluorimetry. Flow cytometry and “cell sorting”. Luminometry.

Enzyme methods and enzyme kinetics: effect of substrate and enzyme concentration, pH and temperature on initial rate. Kinetic parameters. Enzyme inhibitors. Enzyme assays: spectrophotometric, spectrofluorimetric, radioisotope and immunochemical methods. Enzyme assays in Clinical chemistry. Substrate assays: “end-point” and kinetic methods. Enzyme purification: yield and specific activity. Immobilized enzymes and their applications.

Radioisotope techniques: the nature of radioactivity. Types of radioactive decay. Radioactive decay rate and energy. Units of radioactivity. Detection and measurement of radioactivity. Methods based upon excitation; liquid and solid scintillation counting. Determination of counting efficiency. Sample preparation. Methods based upon exposure of photographic emulsion: autoradiography. Receptor-ligand binding: the Scatchard plot.

Immunochemical techniques: production of polyclonal and monoclonal antibodies. Immunoprecipitation in solution.  Immunoelectrophoresis. Immunoblotting. Immunoassays: radioimmunoassays (RIA, IRMA) and enzyme immunoassays (ELISA).

Electrochemical techniques: ion-selective and gas-sensing electrodes. The oxygen electrode and its applications: mitochondrial studies. Biosensors.

 Techniques in Molecolar Biology:

DNA isolation and analysis: nucleic acid electrophoresis, Southern blotting.  Sanger DNA sequencing. Restriction enzymes in the study of genetic diseases: RFLP identification. DNA-binding proteins interaction (foot-printing). "Melting" and annealing of DNA.

RNA analysis: purification of mRNA by affinity chromatography. Northern blotting. RNA microarrays.

Readings/Bibliography

K. Wilson & J. Walker - Principles and Techniques of  Biochemistry  and Molecular Biology - Cambridge University Press 2005

Teaching methods

During the Course the major biochemical techniques will be presented, considering both theoretical and practical aspects. Examples and problems will be discussed with the students.

A short practical class will deal with lab exercises and computer tutorials.

Assessment methods

The final exam is aimed to verify the knowledge of the theoretical bases of the biochemical techniques and the ability to deal with practical problems.

Teaching tools

PC; overhead projector. Biological lab.

Updated slides are available in .pdf format at: http://campus.cib.unibo.it.

Office hours

See the website of Gabriele Hakim