Teacher
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IMPERLINI ESTHER
(syllabus)
Theoretical part (40 hours): - The principles of biochemical research. - Buffer systems. Ionization of amino acids and proteins as a function of pH. - Error theory. Accuracy and precision. - Centrifugation: basic principles of sedimentation; centrifuges and their use; rotors; separation methods in the preparative centrifugation; differential centrifugation; density gradient centrifugation; analytical ultracentrifugation. - Strategies for isolation and purification of proteins; cellular extraction methods; dialysis and ultrafiltration; fractional precipitation with salts, with organic solvents, to the isoelectric point, to heat. - Spectrophotometry: laws and applications; Lambert-Beer law; absorption spectrum of nuclei acids and proteins; spectrophotometric determination of protein concentration: Biuret, Lowry and Bradford methods; enzyme dosing. - Chromatographic methods: adsorption and partition chromatography, affinity, molecular exclusion, ion exchange, hydrophobic interaction, reverse phase liquid chromatography, HPLC. - Electrophoresis: theory and laws; classification of electrophoretic methods; equipment for electrophoresis gel; electrophoresis of agarose, acrylamide and SDS-PAGE; techniques of detection and staining of electrophoretic gels; classical two-dimensional electrophoresis (2-DE) and Differential in Gel Electrophoresis (DIGE technology). - Immunochemical techniques: western blot; immunoprecipitation and immunoaffinity as strategies for the analysis of protein complexes and protein interactions. - Mass spectrometry: principles and application in modern proteomic analysis.
Practical Part (8 hours): - Determination of the protein concentration of an unknown sample using the Bradford method. Spectrophotometer display of absorption spectrum of a protein with/without prosthetic groups; discussion of results.
- Preparation of a polyacrylamide gel in the presence of SDS (SDS-PAGE) and electrophoretic run with pre-stained molecular weight markers, in order to follow the protein mobility during the electrophoretic process. Illustration of devices for the preparation of agarose gel and/or western blotting.
(reference books)
- Wilson K., Walker J. Principles and techniques of practical biochemistry. 5th ed., 2000, Cambridge University Press.
Handouts are provided by the teacher for practical laboratory activities.
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