1. To choose and select appropriate method for extraction of proteins and their subsequent analysis.
2. To apply and utilize electrophoretic techniques for protein investigation (SDS-PAGE, electrophoresis in native conditions, 2-D electrophoresis and Western blotting).
3. To analyse results obtained by above mentioned techniques and to estimate their quality.
4. To compile results obtained by different techniques, to explain them and to carry out conclusions.
5. To edit, organise and present results in the form of scientific article.
6. To edit, organise and present results in the form of oral presentation.
7. To experience team work and learn how to communicate with team members.
It is an introductory course where students get basic principles of protein extraction and learn about techniques for their analyses with emphasis on plant proteins and their post-translational modifications.
1. Extraction of cellular proteins (homogenisation and centrifugation, concentration and purification of extracts)
2. Extraction of extracellular proteins (extraction from nutrient medium, concentration and purification of extracts)
3. Electrophoresis in native conditions (NATIVE PAGE)
4. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
5. Extraction of proteins for two-dimensional electrophoresis (2-DE)
6. 2-DE: 1st dimension - isoelectric focusing (IEF)
7. 2-DE: 2nd dimension - SDS-PAGE
8. Protein visualisation: silver staining and staining with Commassie Brilliant Blue
9. Protein visualisation: fluorescent dyes
10. Western blotting and imunodetection with antibodies
11. Western blotting and glycoprotein detection with lectins
12. Computer programmes for gel analysis
13. Protein analysis by mass spectrometry
14. Protein database
15. Experiment design
1. Protein extraction
2. Bradford protein assay for estimation of protein concentration
3. Protein extracts preparation for SDS-PAGE
4. Protein visualisation by silver staining
5. Protein visualisation by Commassie Brilliant Blue dye
6. Protein visualisation by Sypro Ruby fluorescent dye
7. Analysis of obtained results
8. SDS-PAGE and Western blotting
9. Detection of glycosylated protein by lectins
10. Protein detection by specific antibody
11. Protein extraction for 2-DE
12. Performance of the 1st dimension - isoelectric focusing (IEF)
13. Performance of the 2nd dimension - SDS-PAGE
14. Protein visualisation by Commassie Brilliant Blue dye
15. Gel analysis by computer programme
- Balen, B., Pavoković, D., Peharec Štefanić, P., Krsnik-Rasol, M. Elektroforetske tehnike istraživanja proteina, sveučilišni priručnik, Hrvatska sveučilišna naklada, Zagreb 2011.
- Molecular Biology of the Cell. 5th ed. Alberts, Bruce i sur. New York: Garland Publishing; 2008
- Walker, J.M. The Protein Protocols Handbook, Humana Press, Totowa 1996