KBC/EMB Experimental Methods in Biochemistry

Guarantor: Marek Šebela
Tutorial lecturers: Filip Dyčka, David Kopečný, Lucie Kubienová, Lenka Luhová, Marek Petřivalský, Petr Tarkowski
Exercise: 8 hours/week
Credits: 7
Winter semester
Form of course completion: course credit

Advanced laboratory practicals in biochemistry. Homogenization and extraction of biological material. Precipitation and centrifugation methods. Automated protein assay in serial samples (plate reader). Gel electrophoresis under native conditions, staining for enzyme activity. SDS-PAGE, blotting, immunodetection. Isoelectric focusing. Tricine electrophoresis of proteins. Ion-exchange chromatography of proteins in low-pressure arrangement. Determination of molecular mass of proteins using medium-pressure gel chromatography. Chromatofocusing. HPLC separation of polyamines. In-gel digestion of protein samples and MALDI-TOF peptide mass fingerprinting. Protein intact mass determination using MALDI-TOF mass spectrometry. Localization of proteins and enzymes in tissue sections (light microscopy). Activity assay of oxidases using Clark oxygen electrode. Determination of compounds by amperometric biosensor. Analysis of enzyme interaction with a substrate or inhibitor by absorption spectroscopy. Spectrofluorimetric assay of enzyme activity. Immunochemical methods - Ouchterlony´s double radial immunodiffusion, crossed immunoelectrophoresis, rocket immunoelectrophoresis. Analysis of a glycoprotein using deglycosylating enzymes and gel electrophoresis. Chemical modification of proteins.