Course detail

Laboratory Classes in Biochemistry

FCH-BCO_BCH_PAcad. year: 2018/2019

Fundamentals 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).

Learning outcomes of the course unit

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.

Prerequisites

Examination - BIochemistry I.

Co-requisites

Not applicable.

Recommended optional programme components

Not applicable.

Recommended or required reading

Nelson D.L., Cox M.M. : Lehninger Biochemistry. W.H. Freemann and Co., New York 2007. (CS)
Márová I., Vránová D. : Praktikum z biochemie - pracovní sešit. FCH VUT v Brně, Brno 2002. (CS)
Peč P. a kol. : Laboratorní cvičení z biochemie. FP Universita Palackého, Olomouc 2000. (CS)
Ferenčík M, Škárka B. : Biochemické laboratorne metódy, Alfa, Bratislava 1981. (CS)
Káš J. a kol. : Laboratorní cvičení z biochemie, VŠCHT, Praha 2000. (CS)
Voet D., Voet J.G. : Biochemie. Victoria Publishing, Praha 1990. (CS)

Planned learning activities and teaching methods

The course uses teaching methods in form of Laboratory exercise - 4 teaching hours per week. The e-learning system (LMS Moodle) is available to teachers and students.

Assesment methods and criteria linked to learning outcomes

Participation at 12 experimental exercises and presenting of equivalent reports. Credit test (at minimum 70 %).

Language of instruction

Czech

Work placements

Not applicable.

Course curriculum

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

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.

Specification of controlled education, way of implementation and compensation for absences

Pass out all 12 practices, introduction and test (70%).

Classification of course in study plans

  • Programme BPCP_CHCHT Bachelor's

    branch BPCO_CHMN , 3. year of study, winter semester, 2 credits, compulsory

  • Programme BPCP_CHTP Bachelor's

    branch BPCO_BT , 3. year of study, winter semester, 2 credits, compulsory

  • Programme BKCP_CHTP Bachelor's

    branch BKCO_BT , 3. year of study, winter semester, 2 credits, compulsory
    branch BKCO_PCH , 3. year of study, winter semester, 2 credits, compulsory

  • Programme BPCP_CHTP Bachelor's

    branch BPCO_CHP , 3. year of study, winter semester, 2 credits, compulsory

  • Programme CKCP_CZV lifelong learning

    branch CKCO_CZV , 1. year of study, winter semester, 2 credits, compulsory