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Lecturer(s)
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Šebela Marek, prof. Mgr. Dr.
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Course content
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Introduction. Principles of working with biological material. Homogenization of tissues and extraction of cellular content. Extraction and precipitation methods. Centrifugation methods. Preparative and analytical centrifugation, centrifugation in density gradient. Dialysis. Ultrafiltration. Lyophilization. Chromatographic methods. Adsorption, ion-exchange, gel and affinity chromatography. HPLC and perfusion chromatography. Chromatofocusing. Electromigration methods. Gel electrophoresis. Isoelectric focusing and 2D electrophoresis. Blotting. Capillary electrophoresis. Methods of studying proteins. Assay of protein concentration, amino acid analysis, protein sequencing, peptide mapping, study of post-translational modifications, chemical modifications of proteins, stability studies, chemical synthesis of peptides. Mass spectrometry of proteins, peptide mass fingerprinting, de novo sequencing, characterization of posttranslational modifications, proteins as biomarkers in medicine. Analysis of nucleic acids. DNA mapping by restriction endonucleases, DNA chips, DNA sequencing, polymerase chain reaction (PCR), chemical synthesis of oligonucleotides. Immunochemical methods. Polyclonal and monoclonal antibodies. Immunochemical Immunodiffusion and immunoelectrophoresis. ELISA, immunoblotting. Flow cytometry. Spectrocopic methods. Absorption spectroscopy. Fluorescence spectroscopy. Infrared and Raman spectroscopy. Mösbauer spectroscopy. Chiroptic methods. Resonance spectroscopic methods - NMR a EPR. Protein crystallization. X-ray structural analysis. Electrochemical methods in biochemistry. Polarography and voltammetry. Biosensors. Microcalorimetry. Methods for determination of size and shape of biomacromolecules. Interaction of biomacromolecules with ligands. Study of fast reactions. Buffers, measurement of pH and conductivity. Enzyme analysis. Coupled reactions. Chromogenic and fluorogenic substrates. Radioactivity and its measurements. Autoradiography. Imaging.
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Learning activities and teaching methods
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Lecture
- Preparation for the Exam
- 75 hours per semester
- Attendace
- 39 hours per semester
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Learning outcomes
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Students will acquaint themselves with separation methods and methods of instrumental analysis that are used in current biochemistry.
Students will be able to define main concepts and describe the major experimental methods utilized in biochemical laboratories.
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Prerequisites
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An advanced orientation in topics provided by the subject KBC/BCH Fundamentals of Biochemistry is necessary.
KBC/BCH
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Assessment methods and criteria
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Written exam
Successful passing of written examination - obtaining of at least 50% of credit points. Evaluation: 20 - 18 p. A (1) 18 - 16.p B (1-2) 16 - 14 p. C (2) 14 - 12 p. D (2-3) 12 - 10 p. E (3)
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Recommended literature
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Anzenbacher P., Kovář J. (1986). Metody chemického výzkumu pro biochemiky. MŠ ČSR Praha.
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Bhaskar, A. (2014). Biochemical Methods: A Practical Approach. Alpha Science International Ltd., Oxford.
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Gault, V.A.; McClenaghan, N.H. (2009). Understanding Bioanalytical Chemistry: Principles and Applications. Wiley, Chichester.
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Lottspeich, F.; Engels, J.W. (Eds.). (2018). Bioanalytics: Analytical Methods and Concepts in Biochemistry and Molecular Biology. Wiley-VCH, Weinheim.
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Mikkelsen, S.R.; Cortón, E. (2016). Bioanalytical Chemistry. John Wiley & Sons, Hoboken, NJ, USA.
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Mishra, N. Introduction to Proteomics: Principles and Applications. John Wiley & Sons, Hoboken, NJ, USA. 2010.
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Pingoud, A.; Urbanke, C.; Hoggett J.; Jeltsch, A. (2002). Biochemical Methods : A Concise Guide for Students and Researchers.. Wiley-VCH, Weinheim.
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