Traditional Lessons (up front) (40 hours)

Buffer systems. Ionization of amino acids and proteins as a function of pH. The principles of biochemical research. Error theory. Accuracy and precision. (2 hours)
Centrifugation. Basic principles of sedimentation. Centrifuges and their use. Rotors. Separation methods in the preparative centrifugation. Differential centrifugation. Density gradient centrifugation. Analytical ultracentrifugation. (4 hours)
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. Studies on accessibility of ionizable residues in native proteins. Fluorescence and phosphorescence. (7 hours)
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. (3 hours)
Chromatographic methods: adsorption and partition chromatography, ion exchange, molecular exclusion, affinity, hydrophobic interaction, HPLC, gas chromatography. Efficiency of chromatographic procedures and selection of a convenient method. (6 hours)
Electrophoresis: theory and laws. Classification of electrophoretic methods. Free phase and zonal electrophoresis. Equipment for zonal electrophoresis gel. Gel electrophoresis under native and denaturing conditions (SDS-PAGE). Application of SDS-PAGE. Western blotting. Isoelectrofocusing: principles, equipment and methods. Two-dimensional gel electrophoresis. Capillary electrophoresis. (5 hours)
Studies on the structure of proteins. Determination of amino acid composition, free cysteines, amino acid sequence and disulphide bridges. (4 hours)
Mass spectrometry: principles and application in modern proteomic analysis. (3 hours)
Structural characterization of nucleic acids. PCR. DNA sequencing: automated Sanger method. Southern and Northern blotting. (6 hours)

Laboratory activities (8 hours)

Determination of the protein concentration of an unknown sample using the Bradford method. Construction of the calibration line both manually on millimeter paper and using the least squares method and comparison of the obtained results. Spectrophotometer display of absorption spectrum of a protein with prosthetic groups and one without and discussion of results (4 hours).
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 for nucleic acids and western blotting (4 hours).

Wilson K., Walker J. Principles and techniques of practical biochemistry. 8th ed., 2018, Cambridge University Press.
Handouts are provided by the teacher for practical laboratory activities.