Ateneo - UPO

Measuring biological things.
Techniques for sample preparation in biochemistry
Spectrophotometry.
Chromatography
Protein characterization.
Using antibodies in biotechnology.
Enzymes in diagnostic and research.
Binding assays.

Suggested for student with weak chemistry background:
- Metodologie biochimiche Espressione, purificazione e caratterizzazione delle proteine, Seconda edizione, Zanichelli. A cura di Maria Carmela Bonaccorsi Di Patti, Roberto Contestabile, Martino Luigi Di Salvo
- Metodologie biochimiche e biomolecolari, Strumenti e tecniche per il laboratorio del nuovo millennio, Zanichelli Mauro Maccarrone
- PRINCIPI DI METODOLOGIA BIOCHIMICA di C. De Marco, C. Cini – Ed. Piccin

Wet laboratory exercises aim at acquiring the practical skills required for the use of basic biochemical techniques and for a profitable thesis internship.
The laboratory theory lessons present advanced techniques that the student encounters in the scientific literature.

The student MUST have successfully attended the "safety in chemical and biological laboratories" course.
The knowledge on the structure and properties of molecules provided by inorganic and organic chemistry are preparatory.

The course includes:
- lectures on the main techniques
- computer simulations of the main bioinformatics techniques and exercises
- exercises in chemical-biological didactic laboratories.
Attendance is compulsory.

At the end of the exercises the student will present a laboratory notebook which together with the performance in the laboratory will form the basis for the subsequent evaluation. Please note that the attendance of the didactic workshops is compulsory.

All students who have attended the theoretical lessons and the laboratory (minimum 70%) and performed the exercises on DIR will be declared "suitable".
During the examination of the integrated course the knowledge related to this module will be verified.

Meaning of the measure in the biological field: specificity, accuracy, precision and reproducibility.
Sample preparation techniques for biochemical analyzes (homogenization, lysis with detergents). Extraction with solvents and selective precipitation, their application to the fractionation of biological compounds (extraction of lipids, salting out of proteins and nucleic acids). Centrifugation theory and preparative centrifugation techniques for the fractionation of biological compounds. Use of centrifuges and centrifugal force.
Principles of spectrophotometry. The theoretical bases and the practical aspects of spectroscopic techniques (absorption, fluorescence, polarimetry) and examples of their use for the quantification of biological analytes will be discussed. Characteristics of a spectrophotometer and a fluorimeter, their use.
Radioisotopes in the laboratory: characteristics of radioisotopes of the most common use, their uses in biochemistry and instrumentation used for their quantification.
Principles underlying chromatography, main chromatogical techniques (affinity, gel filtration, ion exchange and reverse phase) and relative matrices. Use of chromatography for the separation of complex mixtures of proteins and lipids based on chemical and physical properties. Instrumentation used (column chromatography, thin layer, HPLC, etc.). Detectors and results analysis (retention coefficients, qualitative and quantitative analyzes).
Principles at the base of electrophoresis with particular attention to fractionation and characterization of proteins. IEF, SDS-PAGE, 2D-PAGE, capillary electrophoresis. Most common detection systems: dyes and western blotting.
Protein characterization: digestion, Edman sequencing, use of mass spectrometry for the characterization of proteins, qualitative and quantitative proteomics with particular attention to mass spectrometry techniques.
Antibody applications in biotechnology, competitive and non-competitive immunological assays, RIA, ELISA, immunofluorescence, western blotting, immunodiffusion and related techniques.
Use of enzymes in diagnostics and research. Characterization of enzymatic kinetics and study of inhibitors. Use of enzymes in clinical diagnostics, kinetic assays and end-points applied to research and diagnostics. Analysis of kinetic parameters of an enzyme.
Binding and binding assays, receptor-ligand equilibrium analysis. Methods for the study of molecular interactions, classical essays with marked molecules, advanced spectrometric methods: Fret, Bret, time resolved fluorescence.

The student will acquire the knowledge of the main biochemical methodologies with particular emphasis on the study of proteins. Such knowledge is useful for the critical reading of technical literature.
The exercises allow you to move fruitfully in the laboratory also in view of the degree internship. For this purpose, the practice in compiling the laboratory notebook is important.