Course code:
404B2
Course name:
Methods in Biochemistry

Academic year:

2024/2025.

Attendance requirements:

(001A2 + 029B2) / 401B2

ECTS:

10

Study level:

basic academic studies

Study program:

Biochemistry: 3. year, winter semester, compulsory course

Teachers:

Zoran M. Vujčić, Ph.D.
full professor, Faculty of Chemistry, Studentski trg 12-16, Beograd

Jelena Z. Radosavljević, Ph.D.
assistant professor, Faculty of Chemistry, Studentski trg 12-16, Beograd

Assistants:

Aleksa D. Savić
junior research assistant, Innovation center, Studentski trg 12-16, Beograd

Nenad M. Drulović

Hours of instruction:

Weekly: three hours of lectures + seven hours of labwork (3+0+7)

Goals:

The main goal of this course is to help students master the basic techniques: the preparation of extracts, proteins and nucleic acids and the determination of their concentration. General chromatographic and FPLC techniques and electrophoresis. Independent processing of the data found in the literature.

Outcome:

Students prepared for independent problem solving in the isolation of proteins, nucleic acids and small molecules, their structural determination and characterization. Training students for their first steps in scientific research.

Teaching methods:

Lectures, experimental classes, tutorials. Students present their work using PowerPoint presentations.

Extracurricular activities:

Coursebooks:

Main coursebooks:

  1. R. Reed, D. Holmes, J. Weyers, A. Jones: Practical skills in biomolecular sciences, 2nd edition, Pearson, Prentice Hall, Harlow, 2003.
  2. Zoran Vujčić: Experimental Biochemistry Practicum, Rantek, 2002.

Supplementary coursebooks:

  1. Robert Scopes: Protein purification, 2000.
  2. D. M. Bollag, D. M. Rozycki, S. J. Edelstein: Protein methods, John Wiley and Sons, 2002.

Additional material:

  Course activities and grading method

Lectures:

0 points (3 hours a week)

Syllabus:

  1. Preparation and stabilization of crude extracts. Sterilization by microfiltration, chaotropes and kosmotropes, buffers.
  2. Biosensors, aptamers. Determination of molecular weights of proteins.
  3. Desalination and concentration of proteins. Protein quantification.
  4. Protein purification: Precipitation and chromatographic methods.
  5. Ion exchange and affinity chromatography.
  6. Ion exchange and affinity chromatography.
  7. EBA, HIC, IMAC.
  8. FPLC - fast protein liquid chromatography.
  9. Electrophoresis and isoelectric focusing (immobilized pH gradient - IPG).
  10. Protein staining techniques, zymograms. Peptide mapping. Chemical modifications of proteins.
  11. Transfer and detection of proteins on membranes. Affinity electrophoresis. 2D electrophoresis.
  12. Preparative electrophoresis. Capillary electrophoresis.
  13. Proteomics - fundamentals. Protein sequencing. MALDI TOF. BLAST.
  14. Isolation, quantification and methods of recombinant DNA. PCR. Expression and purification of recombinant proteins.
  15. NA sequencing.

Labwork:

10 points (7 hours a week)

Syllabus:

Students attend laboratory classes after they have successfully passed an entrance test. If a student does not pass the entrance test and has at least 51% of the total number of points, he has the right to take the entrance test one more time.

Laboratory classes are organized in sets which last for 10 working days.

1-2. Preparation of crude extracts and determination of protein concentration.

3-4. Precipitation methods.

5-6. Matrix preparation, column packing. Determination of the molecular weight of a stained enzyme (HRPO).

7-10. Ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), fast protein liquid chromatography (FPLC): Superdex, HIC, Mono Q.

10-12. Electrophoresis: native and isoelectric focusing, SDS PAGE, UT electrophoresis. 2D electrophoresis.

13-14. DNA. Isolation, quantification and analysis. Restriction digestion. Agarose electrophoresis.

14-15. RNA. Isolation, quantification and analysis.

Written exam:

30 points

Oral exam:

40 points

Tutorial:

20 points