Università Roma Tre

Biochemical Methods Module (Prof. Alessandra di Masi)

- Biochemical methodologies in scientific research. Basic equipment in the biochemistry laboratory (glassware, scales, stirrers, thermostated systems, pH meters, hoods, centrifuges).
- Volumetry and weighing. Preparations of solutions, dilutions, buffer systems.
- Production of recombinant proteins: cloning, mutagenesis, expression systems in bacteria, yeasts, mammalian cells, insect cells. Fusion proteins.
- Methods for protein purification: separation techniques. Extraction of proteins from the biological source. Methods of fractionation of protein mixtures. Homogenization of cells with potter, ultraturrax, sonication, continuous pressure homogenizers (1st Laboratory experience).
- Protein purification with chromatographic techniques: gel filtration, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, hydrophobic interaction chromatography, reverse phase chromatography. HPLC applications (with laboratory experience) (2nd laboratory experience).
- Quantification of proteins. (3rd Laboratory experience)
- Electrophoresis. Polyacrylamide gel electrophoresis in native or denaturing conditions (SDS-PAGE) detection techniques, quantitative densitometric analysis of bands, determination of molecular weight, gradient electrophoresis, preparative electrophoresis, isoelectric focusing, two-dimensional electrophoresis. (4th Laboratory experience)
- Immunochemical techniques: antibodies, western blotting, ELISA and RIA, immunoprecipitation. (5th Laboratory experience)
- Spectroscopic methods for the study of proteins. UV-VIS absorption spectroscopy. Fluorescence spectroscopy. Polarized light spectroscopy. Advanced spectroscopies. (6th Laboratory experience)
- Study of enzymes and proteins. Enzymatic kinetics: calculation of Km, Ki, Ka. Study of the type of inhibition: competitive, non-competitive, uncompetitive and mixed. Use of substrate analogues, effect of pH and temperature (7 ° Laboratory experience).
- Dosage of metabolites. Acid or basic deproteinization. Metabolite assay by spectrophotometric, spectrofluorimetric methods, ATP assay, glucose. Use of HPLC in the dosage of metabolites.
- Biochemical methods for the analysis of molecular interactions. Ligand protein analysis. Protein interaction analysis.

- Keith Wilson and John Walker: Biochimica e biologia molecolare: principi e tecniche. EDs. 2019 Raffaello Cortina.
- M.Maccarrone. Metodologie Biochimiche e Biomolecolari, Ed. Zanichelli Ed.
- Bonaccorsi, Contestabile, Di Salvo. Metodologie biochimiche, Ed. Zanichelli

Slides of each lesson will be made available.