SUBJECT

Gel electrophoresis and its Application in plant physiology

practical

master

Faculty of Science

Biology MSc

4

Semesters 1-4

Autumn/Spring semester

PROTEIN GEL ELECTROPHORESIS

Unit 1: Studies of thylakoid component by gel electrophoresis techniques

Weeks 1 and 2. Lecture: Importance of proteomics, applied techniques. History and principle of gel electrophoresis. Advantages of polyacrylamide gel electrophoresis (PAGE). Composition, chemical structure, and preparation of PAGs. Sample preparation and composition: effects of detergents, reducing compounds, and denaturing agents. Factors influencing the separation of protein bands: physical form of the gel; effective pore size; shape, size and charge density of proteins (Fergusson plot analysis); dissociating/non-dissociating, continuous/discontinuous (multiphasic) buffer system, choice of pH and running conditions. PAGE techniques: application of native, blue-native, SDS, gradient, 2D PAGE. Protein staining and quantification: Coomassie-, Ag-, and fluorescent dyes, autoradiography. Characterization and identification of proteins: determination of molecular weight, peptide mapping, Western blotting, N-terminal sequencing, mass spectrometry. Practical: Separation of chlorophyll-proteins by native PAGE. Gel preparation, sample preparation and loading, running of the gels. Scanning of gels and evaluation by Phoretix software. Preparation and running of denaturing SDS gels. Western blotting. Evaluation of blots.

Weeks 3 and 4. Lecture: Theory of isoelectric focusing (IEF). Native and denaturing IEF. Application of IEF and equipments. Chemicals used for IEF. Methods and equipments for IEF gel preparation. Sample preparation for IEF: isolation, delipidation methods. Solubilization, properties of detergents. Running conditions for IEF gels. Staining procedures and evaluation methods in IEF. Preparation of IEF gels for 2D electrophoresis. Methods and equipments used for 2D electrophoresis. Staining and evaluation of 2D gel slabs. Practical: Gel preparation for IEF. Sample preparation for IEF: isolation, delipidation, and solubilization. Assembling of IEF equipment. Sample loading. Running of gels.

Weeks 5 and 6. Practical: Staining (Coomassie) and evaluation of IEF gels. Preparation of IEF gels for 2D electrophoresis. Sample loading and running of gels. Staining (Ag) and evaluation of 2D gel slabs.

Unit 2: Studies of isoenzyme activities by gradient PAGE

Week 7. Lecture: Plants under stress, protective enzymes. Characterization of superoxide dismutase (SOD) and peroxidase (POD) isoenzymes, the importance of tracking their activity changes in plant stress physiology. Gradient PAGE: properties and advantages. The advantage of modified native PAGE. Detecting isoenzyme activities: the interpretation of activity, density-activity relationship, positive and negative staining methods. Practical: Pouring slab gradient gels. Meantime, demonstration of the evaluation of activity stained electrophoretograms by the Phoretix 1D software.

Week 8. Practical: Preparing izoenzymes from control and stressed plant tissues. Determination of protein content. Sample preparation. Running the gel. Detection of enzymes by activity staining: POD – positive, SOD – negative staining method. Gel scanning and evaluation.

NUCLEIC ACID GEL ELECTROPHORESIS

Unit 3: Agarose and polyacrylamide gel electrophoresis of nucleic acids

Week 9. Lecture: Application of agarose GE for general and special purposes (low melting – LM, low gelling). Factors influencing the rate of speed of DNA: size and conformation of DNA, agarose concentration, applied voltage, buffer composition, presence of stain. Theory and practice of pulsed-field GE. Separation limit. Effects of electric field: pulse length and ratio, strength and angle of the field. Techniques: Field Inversion GE, Transverse Alternating Field Electrophoresis, Rotating GE (Contour- clamped, Homogeneous Electric Field). Extraction of DNA from the agarose gel: electroelution, LM agarose, agarase, centrifugation, commercially available kits (DNA binding, washing, and elution). Theory and practice of Southern- and Northern-blots. Possibilities for DNA labelling. Methods and application of polyacrylamide gel electrophoresis. Preparation of gels, applied voltage, detection methods.

Weeks 10 to 12.: Practical: Agarose gel electrophoresis. Extraction of DNA from plant tissues by two different methods. Quantification and characterization of the extracted DNA. Digestion by restriction enzymes. Separation of fragments on agarose gel. Blotting onto cellulose membrane. DNA staining on the membrane.

Weeks 13 and 14. Practical: DNA polyacrylamide gel electrophoresis. Gel preparation. Sample preparation. Sample separation. Staining of DNA bands.

• Gel Electrophoresis of Proteins (BD Hames ed.), OUP Oxford, 1998 1998, ISBN 9780191565632,

• Gel Electrophoresis of Nucleic Acids (BD Hames & D Rickwood, eds.) ISBN 0-19-963083-6, 1990, Oxford Univ. Press, Oxford, England