Setting up a laboratory and basic equipment for biochemical research. Buffers and reagents necessary for working with proteins and protein purification. Protein isolation from raw biological material, methods of lysis and disruption of various cell and tissue types. Maintaining protein stability during isolation, handling and purification. Buffer and solvent exchange using semipermeable membranes: dialysis and ultrafiltration. Protein purification and concentration by precipitation using ammonium sulphate, polyethylene glycol, organic solvents and trichloroacetic acid. Chromatographic techniques of protein separation and purification: ion-exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography. Affinity chromatography: conventional methods of affinity chromatography and affinity chromatography of tagged recombinant proteins. Affinity chromatography of glycoproteins and immunoglobulins. Use of protein tags in biochemistry, in vivo and in vitro protein tagging and labelling. Fluorescent labelling, covalent modification and protein immobilization.
High pressure and high performance liquid chromatography, properties of chromatography resins and instrumentation for high performance protein purification.
Theoretical fundaments of electrophoresis and electrophoretic protein separations. Native and denaturing electrophoresis. SDS-PAGE, isoelectric focusing and 2D-PAGE. Detection of macromolecules after electrophoretic separation. Western blot. Capillary electrophoresis.
Protein analysis: determination of protein concentration and enzymatic activity. Detection and quantification of impurities in protein samples. Analysis of structural heterogeneity. Analysis of posttranslational modifications. Target enrichment. Special considerations for handling of membrane proteins, macromolecular and ribonucleoprotein complexes, supramolecular structures.
- OBVEZNA LITERATURA:
R. R. Burgess and M. P. Deutscher (ed.): Guide to Protein Purification, 2nd Edition. Methods in Enzymology, (2009) 463:1-851
R. K. Scopes: Protein Purification: Principles and Practice, 3rd edition. Springer-Verlag, New York (1994)
G T. Hermanson: Bioconjugate Techniques, 2nd edition. Academic Press, London (2008)
Recently published scientific articles
- DODATNA LITERATURA:
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