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Course, academic year 2023/2024
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Biochemical methods - MC250P09A
Title: Metody biochemie
Czech title: Metody biochemie
Guaranteed by: Department of Biochemistry (31-250)
Faculty: Faculty of Science
Actual: from 2021 to 2023
Semester: summer
E-Credits: 3
Examination process: summer s.:
Hours per week, examination: summer s.:2/0, Ex [HT]
Capacity: unlimited
Min. number of students: unlimited
4EU+: no
Virtual mobility / capacity: no
State of the course: taught
Language: Czech
Note: enabled for web enrollment
Guarantor: doc. RNDr. Miroslav Šulc, Ph.D.
Teacher(s): prof. RNDr. Jiří Hudeček, CSc.
doc. RNDr. Miroslav Šulc, Ph.D.
Annotation -
Introduction to the principles and use of the methods of biochemical research: chemical and biomedical informational resources, evaluation and presentation of results, planning and documentation of experiments, scientific ethics. Organization and equipment of a biochemical laboratory, characteristics of various biological source materials. Basic techniques for isolation of biomolecules: precipitation, salting-out, ultrafiltration and dialysis, centrifugation. Chromatographic and electromigration methods. Immunochemical detection and analysis. Legal conditions for the work with laboratory animals, principles of their use. Work with isolated organs, tissues, and cells. Elements of microscopy - optical and electronic. Analytical use of colorimetry and (UV-VIS) absorption spectrometry. Potentiometry and voltammetry, pH, biosensors.

Lectures - in Czech, English on request
Last update: Šulc Miroslav, doc. RNDr., Ph.D. (14.02.2023)
Literature -

Recommended literature:

In Czech:
Anzenbacher P, Kovář J: Metody chemického výzkumu pro biochemiky, MŠ ČSR Praha, 1986.

Kalous V et al.: Metody chemického výzkumu, SNTL Praha, 1987, 431 str.

Prosser V et al.: Experimentální metody biofyziky, Academia Praha, 1989, 712 str.

M. Kodíček, V. Karpenko: Biofzsikální chemie, Academia Praha, 2000

plus literature dealing with individual methods, and groups of methods.

 

In English:

A. G. Marshall: Biophysical Chemistry. J. Wiley 1978

C. R. Cantor, P. R. Schimmel: Biophysical Chemistry. (in particular Part. II) WH Freeman and Co, 1980 (with many new printings published later)

and the numerous newer books on the subject, but these two are really the best ones (real classics)  for an introductory reading.

Last update: Hudeček Jiří, prof. RNDr., CSc. (01.10.2020)
Requirements to the exam -
The written exam consists of two parts/questions (draw lots): 1st simple calculation (linked to practical tasks in biochemical practices), 2nd teoretical parts consisting 4 questions: two from the subject matter ("bigger topic" - 1-2 lectures), two questions from the lectured topic ("minor topic" - sub-question in the scope of subchapter). The condition for passing the test is a successful solution of part 1 and minimally 60% evaluation of teoretical part.
Last update: Šulc Miroslav, doc. RNDr., Ph.D. (23.02.2024)
Syllabus -

I. CHEMICAL INFORMATICS

Primary sources - scientific papers, technical reports, dissertations, conference proceedings, patents. Peer-review process. Secondary sources - bibliographic, factographic information. Abstract and signal journals, current awareness, computer bibligraphic databases. Chemical Abstracts, Web of Science, Current Contents. Specialized abstract services for biomedical sciences, MEDLINE. Biochemical databases of protein and NA sequences, structures.

II. ANALYSIS AND PRESENTATION OF RESULTS

Observation and experiment. Elements of statistics and theory of measurement. Significant digits. Graphic methods for evaluation of results, linear and nonlinear regression.Nomograms.Writing iof grant applications, laboratory reports, preparation for oral presentation and thesis defense, preparation of posters and scientific papers. Scientific ethics.

III. BASIC TECHNIQUES IN BIOCHEMICAL LABORATORY

Specific features of biomaterials and of the work in biochemical laboratories, basic equipment (consumables, tubing, biocompatibility, semimicroscale work). Separation and purification of biomolecules - planning, follow-up, specific content/activity. Basic assays (protein, NA).Auxilliary substances - water (distiled, deionised, reverse osmosis), buffers in biochemistry. Disintegration and mechanical sorting (homogenizers, mixers and mills, mechanical sieves, centrifugation), solubilisation. Extraction (Soxhlet extractor). Salting-in and salting-out, Hoffmeister series, precipitation. Preparative centrifugation (isopycnic, fractional. Dialysis and ultrafiltration, evaporation, free-drying, storage of samples and preparations.

IV. CHROMATOGRAPHIC SEPARATION

Basic principles of the method and its theory. Overview of methods, their mobile and stationary phases, separation principles. Gradient and isocratic method, frontal, zonal and selectiver elution. Countercurrent extraction and distillation as models.Droplet countercurrent chromatography. GC (detector function), paper and thin-layer chromatography (TLC, HPTLC). SFC. Liquid chromatography, HPLC and perfusion chromatography. Technical elements and metodology. HPLC, size-exclusion, distribution, adsorption, ionex and chelatation chromatography, chropmatofocusation, affinity chromatography.Field flow fractionation.

V. ELECTROMIGRATION

Principles of the methods - electrophoresis, isoelectric focusing, isotachoforesis.
Free and zonal electrophoresis - stabilisation against convection and diffusion. SDS/PAGE, agarose gel elfo, capillary techniques.Affinity elfo, electrophoresis in denaturating conditions.
Blotting and two-dimensional techniques.

VI. IMMUNOCHEMICAL METHODS
Immunoreactivity (basic terms an dconcepts), antibody preparation. Agglutionation and precipitation, titre. Immunodiffusion, immunoelectrophoresis. Immunoassays and immunometric methods (competitive, non-competitive, solid-phase and homogeneous methods).

VII. SPECTROPHOTOMETRY AND COLORIMETRY

Elementary theory (Lambert-Beer law, analytical use of spectra, frequency, wavenumber and wavelength, Subjective and objective photometry. Spectral measurements, principles of instrument construction and use. Typical biomolecular chromophores.

VIII. ELECTROANALYTICAL METHODS

Principles and overview of the electroanalytical methods. Potentiometry, pH, enzyme electrodes. Voltammetry, oxygen electrode, polarography.. Conductometry.

IX. EXPERIMENTS WITH LIVING MATERIAL

Laboratory animals, ethical an legal aspects, alternatives, transgenic animals. Legal aspects of experiments wit animal and human material. Isolated organs, tissue and cell cultures.

X. MICROSCOPY

Optical microscopy - principles, physical and chemical methods of increasing the optical contrast of objects. Electron microscopy (trasmission, scanning). Atomic field microscopy.
Microscopy and chemical analysis.

XI. UV-VIS SPECTROSCOPY

-electron absorption molecular spectra

-UV-VIS spektroscopy of proteins, nucleic acids (photometric assayes) and small bio-molecules - chromogenuc substrates

-derivational and differece spectra UV-VIS

Last update: Šulc Miroslav, doc. RNDr., Ph.D. (09.02.2023)
 
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