Course detail

Laboratory Classes in Biochemistry

FCH-BAO_BCH_PAcad. year: 2024/2025

FFundamentals of biochemical analytics and separative methods. Methods of the determination of proteins. Enzymes, enzyme kinetics. Isolation and purification of nucleic acids. Specific methods : isolation of enzymes from various types of biological material, protein chromatograpgy, electophoretic techniques (PAGE-SDS, electrophoresis in agarose gel).

Language of instruction

English

Number of ECTS credits

2

Mode of study

Not applicable.

Guarantor

prof. RNDr. Ivana Márová, CSc.

Department

Institute of Food Science and Biotechnology (ÚCHPBT)

Offered to foreign students

The home faculty only

Entry knowledge

Examination - Biochemistry I.

Rules for evaluation and completion of the course

Participation at 12 experimental exercises and presenting of equivalent reports. Credit test (at minimum 70 %).
Pass out all 12 practices, introduction and test.

Aims

The course is focused on some methods of manipulation with biological material, basic biochemical separation as well as analytical techniques and basic processes using in enzymology. The knowledge obtained after passing the course can serve as the basis of following practical courses, especially practices on biotechnology and gene technologies.
Students will obtain knowledge about isolation and purification of biopolymers and other components present in various types of biological material. Further, stability of biomolecules, and methods of their characterization, analysis and applications will be demonstrated.

Study aids

Not applicable.

Prerequisites and corequisites

Not applicable.

Basic literature

Márová I., Vránová D./Pracovní sešit - praktikum z biochemie (EN)

Recommended reading

Not applicable.

Elearning

eLearning: currently opened course

Classification of course in study plans

  • Programme NPAP_ENVI Master's 1 year of study, winter semester, elective

Type of course unit

 

Laboratory exercise

52 hod., compulsory

Teacher / Lecturer

Ing. Jiří Holub, Ph.D.

Syllabus

1. Saccharides
a) qualitative analysis of saccharides
b) thin-layer chromatography of saccharides

2. Lipids
a) preparation of lipid fractions form egg yolk
b) enzyme hydrolysis of lecithin using phospholipase D
c) identification of egg lipids

3. Lipid-soluble pigments
a) adsorption chromatography of chlorpohyll pigments (spinach) and carotenoids (paprika) by TLC, isolation of beta-carotene fraction
b) isolation of beta-carotene from paprika by column adsorption chromatography
c) RP-HPLC of beta-carotene fractions isolated from TLC and adsorption chromatography
d) spectrophotometric analysis of isolated pigments

4. Amino acids
a) chemical reactions of amino acids and peptides
b) separation of amino acid mixture by paper chromatography

5. Proteins
a) analysis of protein concentration by biuret method
b) analysis of protein concentration by Hartree-Lowry method
c) analysis of protein concentration by Bradford method ( Coomassie blue)
d) comparison of methods used for quantitative analysis of protein concentration

6. Enzymes I
a) determination of enzyme activity of amino transferases isolated from pig heart
b) determination of initial velocity of enzyme reaction (hydrolysis of gelatin by trypsin)

7. Enzymes II - kinetics
a) substrate specificity of saccharase
b) pH optimum of enzyme reaction catalysed by alpha-amylase

8. Isolation and purification of nucleic acids from different natural sources
a) isolation of DNA from calf thymus
b) isolation of RNA from yeasts
c) identification of DNA and RNA by reaction with diphenyl ammine

9. Isolation and partial purification of enzymes frionm several types of biological material
a) isolation of ammin oxidase from pea by fraction precipitation; dialysis
b) analysis of purity of crude enzyme preparatives; isolation scheme
c) isolation of urease from soybean; application for analysis of urea concentration

10. Gel permeation chromatrography
a) preparation of azo-derivative of albumin
b) Sephadex gel preparation, column stabilization; determination of death volume
c) isolation of colour protein derivative by gel filtration using Sepgadex G-25

11. Separation of proteins and nucleic acids by horizontal electrophoresis
a) electrophoresis of protein mixture on cellulose acetate
b) electrophoresis of nucleic acids in agarose gel

12. Determination of relative molecular mass of proteins by PAGE-SDS electrophoresis
a) preparation of polyacrylamide gel and vertical electrophoresis apparatus
b) determination of Mr of selected proteins by PAGE-SDS electrophoresis

Elearning

eLearning: currently opened course